Process for the determination of peptides corresponding to immunologically important epitopes and their use in a process for determination of antibodies or biotinylated peptides corresponding to immunologically important epitopes, a process for preparing them and compositions containing them

ABSTRACT

The technical problem underlying the present invention is to provide peptides corresponding to immunologically important epitopes on bacterial and viral proteins, as well as the use of said peptides in diagnostic or immunogenic compositions. The invention relates to a process for the in vitro determination of antibodies, wherein the peptides used are biotinylated, particularly in the form of complexes of streptavidin-biotinylated peptides or of avidin-biotinylated peptides.

This application is a divisional application of Ser. No. 10/621,675,filed Jul. 18, 2003 (pending), which is a divisional of application Ser.No. 09/576,824, filed May 23, 2000 (now U.S. Pat. No. 6,667,387), whichis a divisional of application Ser. No. 08/723,425, filed Sep. 30, 1996,which is a divisional of application Ser. No. 08/146,028, filed Nov. 22,1993, which is a 371 application of PCT/EP93/00517, which claims benefitof EP 92400598.6, filed Mar. 6, 1992, the entire content of each ofwhich is hereby incorporated by reference in this application.

DETAILS

The technical problem underlying the present invention ell is to providepeptides corresponding to immunologically important epitopes onbacterial and viral proteins, as well as the use of said peptides indiagnostic or immunogenic compositions.

Recent developments in genetic engineering as well as the chemistry ofsolid phase peptide synthesis have led to the increasingly wider use ofsynthetic peptides in biochemistry and immunology. Protein sequences,which become available as a result of molecular cloning techniques canbe synthesized chemically in large quantities for structural,functional, and immunological studies. Peptides corresponding toimmunologically important epitopes found on viral and bacterial proteinshave also proven to be highly specific reagents, which can be used forantibody detection and the diagnosis of infection.

Despite the many advantages synthetic peptides offer, there are a numberof disadvantages associated with their use. Because of their relativelyshort size (generally less than 50 amino acids in length), theirstructure may fluctuate between many different conformations in theabsence of the stabilizing influence of intramolecular interactionspresent in the full-length protein.

Furthermore, the small size of these peptides means that their chemicalproperties and solubilities will frequently be quite different fromthose of the full-length protein and that the contribution of individualamino acids in the peptide sequence toward determining the overallchemical properties of the peptide will be proportionally greater.

Many immunological assays require that the antigen used for antibodydetection be immobilized on a solid support. Most enzyme-linkedimmunosorbent assays (ELISA) make use of polystyrene as the solid phase.

Many proteins can be stably adsorbed to the solid phase and presentsequences, which are accessible for subsequent interactions withantibodies. Because of their small size, direct adsorption of peptidesto the solid phase frequently gives rise to unsatisfactory results forany of a number of reasons.

Firstly, the peptide may not possess the correct overall charge or aminoacid composition, which would enable the peptide to bind to the solidphase. Secondly, the same amino acid residues, which are required forbinding to the solid phase, may also be required for antibodyrecognition and therefore not available for antibody binding. Thirdly,the peptide may become fixed in an unfavourable conformation uponbinding to the solid phase, which renders it unrecognizable to antibodymolecules. In many cases, it is neither possible nor necessary todistinguish between these possibilities. Binding to the solid phase canbe increased and made less sensitive to the specific chemical propertiesof a peptide by first coupling the peptide to a large carrier molecule.Typically, the carrier molecule is a protein.

While the amount of peptide bound to the solid phase, albeit indirectly,can in some cases be increased by this method, this approach suffersfrom the fact that the linkage between the peptide and the carrierprotein frequently involves the side chains of internal trifunctionalamino acids whose integrity may be indispensable for recognition byantibodies. The binding avidity of antisera for the internally modifiedpeptide is frequently very much reduced relative to the unmodifiedpeptide or the native protein.

The production of antisera to synthetic peptides also requires in mostcases that the peptide be coupled to a carrier. Again, the couplingreaction between an internal trifunctional amino acid of the peptide andthe carrier is likely to alter the immunogenic properties of thepeptide.

There exist many methods for performing coupling reactions and most ofthe procedures in current use are discussed in detail in VanRegenmortel, M. H. V., Briand, J. P., Muller, S., and Plaud, S.;Laboratory Techniques in Biochemistry, and Molecular Biology, vol. 19,Synthetic Polypeptides as Antigens, Elsevier Press, Amsterdam, N.Y.,Oxford, 1988. In addition to these procedures, unprotected peptides canalso be biotinylated using commercially available reagents such asN-hydroxysuccinimidobiotin or biotinamidocaproate N-hydroxysuccinimideester. Many of these reagents are discussed in Billingsley, M. L.,Pennypacker, K. R., Hoover, C. G., and Kincaid, R. L., Biotechniques(1987) 5(1): 22-31. Biotinylated peptides are capable of being bound bythe proteins streptavidin and avidin, two proteins, which exhibitextraordinarily high affinity binding to biotin.

In certain instances, it is possible to selectively couple biotin to anunprotected peptide or an unprotected peptide to a carrier. This may beaccomplished by synthesizing the peptide with an additionaltrifunctional amino acid added to one of the ends, which is capable ofparticipating in the coupling reaction. This approach will only besuccessful, however, as long as this amino acid is not a criticalresidue in the immunogenic sequence of interest and as long as thecoupling agent chosen is sufficiently selective. No single technique isapplicable to all unprotected peptides regardless of their amino acidcomposition.

The etiological agent responsible for non-A, non-B hepatitis has beenidentified and termed hepatitis C virus (HCV). Patent applicationEP-A-0318216 discloses sequences corresponding to approximately 80% ofthe viral genome. The availability of these sequences rapidly led to theelucidation of the remainder of the coding sequences, particularly,those located in the 51 end of the genome (Okamoto; J. Exp. Med. 60,167-177,1990). The HCV genome is a linear, positive-stranded RNAmolecule with a length of approximately 9400 nucleotides. With theexception of rather short untranslated regions at the termini, thegenome consists of one large, uninterrupted, open reading frame encodinga polyprotein of approximately 3000 amino acids. This polyprotein hasbeen shown to be cleaved co-translationally into individual viralstructural and non-structural (NS) regions. The structural proteinregion is further divided into capsid (Core) and envelope (E1 and E2proteins. The NS regions are divided into NS-1 to NS-5 regions.

A number of independent patent applications have employed a variety ofstrategies to determine the locations of diagnostically important aminoacid sequences and many of these studies have led to the identificationof similar regions of the HCV polyprotein.

The NS4 region has mainly been studied in EP-A-0 318 216, EP-A-0 442394, U.S. Pat. No. 5,106,726, EP-A-0 489 986, EP-A-0 484 787, and EP-A-0445 801. Unfortunately only 70% of HCV-infected individuals produceantibodies to NS4, neither the synthetic nor recombinant proteinscontaining sequences from this region are adequate for identifying allinfected serum samples. The nucleocapsid or Core region has been studiedin patent applications EP-A-0 442 394, U.S. Pat. No. 5,106,726, EP-A-0489 986, EP-A-0 445 801, EP-A-0 451 891 and EP-A-0 479 376. It was foundthat these peptides often used as mixtures were more frequentlyrecognized by antibodies (85-90%) in sera from chronically infectedindividuals than were the peptides derived from NS4. The NS5 region wasstudied in patent applications EP-A-0 489 986 and EP-A-0 468 527.Depending on the serum panel used, more than 60% of NANB hepatitis canbe shown to contain antibodies directed against these peptides. The NS3region was also studied in patent application EP-A-0 468527. Allavailable evidence suggests that the most dominant epitope of NS3 arediscontinuous in nature and cannot be adequately represented bysynthetic peptides. The E1 region, which is potentially interesting as aregion from the outer surface of the virus particles (possibleimmunogenic epitopes), was studied in both patent applicationsEP-A:-0468 527 and EP-A-0507 615. The E2/NSI region was studied for thesame reason as E1. Comparisons of this region from different HCVvariants elucidated that this protein contains variable region which arereminiscent of the HIV V3 loop region of gp120 envelope protein. Fourpeptides were found in EP-A 0468 527 which were shown to containrelatively infrequently recognized epitopes. Finally, the NS2 region ofHCV was analyzed in EP-A-0 486 527. However, the diagnostic value ofthis region is not clear yet. Virtually all patent applicationsconcerning diagnostically useful synthetic peptides for antibodydetection describe preferred combinations of peptides. Most of theseinclude peptides from the HCV core protein and NS4. In some cases,peptides from NS5 (EP-A-0 489 968 and EP-A-0 468 527), and E1 and E2/NS1are included (EP-A-0 507 615 and EO-A-0 468 527).

Different patent applications have addressed the problem of findingdiagnostically useful epitopes of human immunodeficiency virus (HIV). Animportant immunodominant region containing cyclic HIV-1 and HIV-2peptides was found in patent application EP-A-0 326 490. In EP-A-0 379949, this region was asserted to be even more reactive with HIV-specificantibodies in case a biotin molecule was coupled to these cyclic HIVpeptides. SU-A-161 22 64 also describes the use of a biotinylatedpeptide in a solid phase immunoassay for the detection of HIVantibodies. Other applications have looked for useful HIV epitopes inthe hypervariable, V3 loop region of gp120 (such as EP-A-0 448 095 andEP-A-0 438 332). U.S. Pat. No. 4,833,071 provides peptide compositionsfor detection of HTLV I antibodies.

Deciding whether or not an epitope is diagnostically useful is notalways straightforward and depends to an extent on the specificconfiguration of the test into which it is incorporated. It should beideally an immunodominant epitope, which is recognized by a largepercentage of true positive sera or should be able to complement otherantigens in the test to increase the detection rate.

Epitopes which are not frequently recognized may or may not bediagnostically useful depending on the contribution they make towardsincreasing the detection rate of antibodies in true positive sera andthe extent to which incorporation of these epitopes has an adverseeffect on the sensitivity of the test due to dilution of other strongerepitopes.

Peptides can thus be used to identify regions of proteins, which arespecifically recognized by antibodies produced as a result of infectionor immunization. In general, there are two strategies which can befollowed. One of these strategies has been described by Geysen, H. M.,Meloen, R. H. and Barteling, S. J.; Proc. Natl. Acad. Sci. USA (1984)81:3998-4002. This approach involves the synthesis of a large series ofshort, overlapping peptides on polyethylene rods derivatized with anoncleavable linker such that the entire length of the protein orprotein fragment of interest is represented. The rods are incubated withantisera and antibody binding is detected using an anti-immunoglobulin:enzyme conjugate. A positive reaction immediately identifies thelocation and sequence of epitopes present in the protein sequence. Thistechnique has the advantage that all peptides are uniformly linked tothe solid support through their carboxy-terminus. While this methodallows for very accurate mapping of linear epitopes, the length of thepeptides, which can be reliably synthesized on the rods, is limited.This may sometimes present problems if the length of the epitope exceedsthe length of the peptides synthesized.

A second approach to epitope mapping involves the synthesis of largerpeptides, generally between fifteen and thirty amino acids in length,along the sequence of the protein to be analyzed. Consecutive peptidesmay be contiguous but are preferably overlapping. Following cleavage,the evaluation of antibody binding to the individual peptides isassessed and the approximate positions of the epitopes can beidentified. An example of this approach is given in Neurath, A. R.,Strick, N., and Lee, E. S. Y.; J. Gen. Virol. (1990) 71:85-95. Thisapproach has the advantage that longer peptides can be synthesized whichpresumably more closely, resemble the homologous sequence in the nativeprotein and which offer better targets for antibody binding. Thedisadvantage of this approach is that each peptide is chemically uniqueand that the conditions under which each peptide can be optimally coatedonto a solid phase for immunological evaluation may vary widely in termsof such factors as pH, ionic strength, and buffer composition. Thequantity of peptide which can be adsorbed onto the solid phase is alsoan uncontrolled factor which is unique for each peptide.

The main purpose of the present invention is to provide modifiedpeptides corresponding to immunologically useful epitopes with saidmodified peptides having superior immunological properties overnon-modified versions of these peptides.

Another aim of the present invention is to provide modified peptidescorresponding to immunologically useful epitopes, which could not beidentified through classical epitope mapping techniques.

Another aim of the present invention is to provide a process for the invitro determination of antibodies using said peptides, with said processbeing easy to perform and amenable to standardization.

Another aim of the invention is to provide a process for thedetermination of peptides corresponding to immunologically importantepitopes on bacterial and viral proteins.

Another aim of the invention is to provide a method for preparingprotein sequences used in any of said methods. Another aim of theinvention is to provide a method for preparing protein sequence, whichcan be used in a process for the determination of their epitopes or inan in vitro method for the determination of antibodies.

Another aim of the invention is to provide intermediary compounds usefulfor the preparation of peptides used in the above-mentioned methods.

Another aim of the present invention is also to provide compositionscontaining peptides determined to correspond to immunologicallyimportant epitopes on proteins for diagnostic purposes.

Another aim of the present invention is also to provide compositionscontaining peptides determined to correspond to immunologicallyimportant epitopes on proteins for vaccine purposes.

According to the present invention, a series of biotinylated peptidesrepresenting immunologically important regions of viral proteins havebeen identified and prepared by solid phase peptide synthesis. Thesepeptides have been identified to be very useful for (i) the detection ofantibodies to HCV, and/or HIV, and/or HTLV-I or II. In some preferredarrangements, these peptides were also found or are at least expected,to be useful in stimulating the production of antibodies to HCV, and/orHIV, and/or HTLV-I or II in healthy animals such as BALB/C mice, and ina vaccine composition to prevent HCV and/or HIV, and/or HTLV-I or IIinfection.

As demonstrated in the Examples section of the present invention, theuse of biotinylated peptides also allows the determination ofimmunologically important epitopes within a previously determinedprotein sequence. The determination of immunologically importantepitopes using non-biotinylated peptides, which are covalently coupledto the solid phase, often fails to localize these epitopes.

Especially in case of localization of structural epitopes, the use ofbiotinylated peptides seems to be quite successful.

(1) According to the present invention, a peptide composition useful forthe detection of antibodies to HCV, and/or HIV, and/or HTLV-I or IIcomprise peptides corresponding to immunologically important epitopesbeing of the structure:(A)-(B)—(X)—(Y)-[amino acids]_(n)-Y—(X)-Zwhere

-   -   [amino acids]_(n) is meant to designate the length of the        peptide chain where n is the number of residues, being an        integer from about 4 to about 50, preferably less than about 35,        more preferably less than about 30, and advantageously from        about 4 to about 25;    -   B represents biotin;    -   X represents a biotinylated compound, which is incorporated        during the synthetic process;    -   Y represents a covalent bond or one or more chemical entities        which singly or together form. a linker arm separating the amino        acids of the peptide proper from the biotinyl moiety B or X, the        function of which is to minimize steric hindrance which may        interfere with the binding of the biotinyl moiety B or X to        avidin or streptavidin wherein Y is not a covalent bond, it is        advantageously at least one chemical entity and may consist of        as many as 30 chemical entities but will consist most frequently        of 1 to 10 chemical entities, which may be identical or        different, more preferably glycine residues, β-alanine,        4-aminobutyric acid, 5-aminovaleric acid, or 6-aminohexanoic        acid;    -   B and X being enclosed in parentheses to indicate that the        presence of biotin or a biotinylated compound in these positions        is optional, the only stipulation being that B or X be present        in at least one of the positions shown;    -   A, when present, as indicated by parentheses, represents (an)        amino acid(s), an amino group, or a chemical modification of the        amino terminus of the peptide chain;    -   Z represents (an) amino acid(s), an OH-group, an NH2-group, or a        linkage involving either of these two chemical groups wherein        the amino acids are selectively chosen to be immunodominant        epitopes which are recognized by a large percentage of true        positive sera or are able to complement other antigens in the        test to increase the detection rate and B interacts with the        selected amino acids to produce a compound with greater        diagnostic sensitivity.

The peptide composition comprises at least one and preferably acombination of two, three, four or more biotinylated peptides chosenfrom the following sequences: 1. Human immunodeficiency Virus type IEnvelope Peptides: a. gp41 1. gp41, isolate HTLV-IIIB(A)-(B)-(X)-Y-Ile-Trp-Gly-Cys-Ser- (SEQ ID NO:1) Gly-Lys-Ile-Cys-Y-(X)-Z2. (A)-(B)-X)-Y-Ile-Trp-Gly-Cys-Ser- (SEQ ID NO:2)Gly-Lys-Leu-Ile-Cys-Thr-Thr-Ala-Val- Pro-Trp-Asn-Ala-Ser-Y-(X)-Z 3.(A)-(B)-(X)-Y-Glu-Arg-Tyr-Leu-Lys- (SEQ ID NO:3)Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly- Cys-Ser-Gly-Lys-Leu-Ile-Y-(X)-Z 4.(A)-(B)-(X)-Y-Leu-Gln-Ala-Arg-Ile- (SEQ ID NO:4)Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp- Gln-Gln-Leu-Y-(X)-Z 5. gp41,isolate Ant70 (A)-(B)-(X)-Y-Leu-Trp-Gly-Cys-Lys- (SEQ ID NO:5)Gly-Lys-Leu-Val-Cys-Y-(X)-Z 6. gp41, isolate ELI(A)-(B)-(X)-Y-Asp-Gln-Gln-Leu-Leu- (SEQ ID NO:6)Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-His-Ile-Cys-Thr-Thr-Asn-Val-Pro-Trp-Asn- Y-(X)-Z b. gp 120 1. Partial V3loop sequence, consensus (A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys- (SEQ IDNO:7) Ser-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-Ile-Ile-Gly- Y-(X)-Z 1. a. Complete V3 loopsequence, consensus (A)-(B)-(X)-Y-Cys-Thr-Arg-Pro-Asn- (SEQ ID NO:8)Asn-Asn-Thr-Arg-Lys-Ser-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-Ile-Ile-Gly-Asp-Ile-Arg-Gln- Ala-His-Cys-Y-(X)-Z 2. Partial V3loop sequence, isolate HIV-1 SF2 (A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys- (SEQID NO:9) Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Ile-Gly- Y-(X)-Z 3. Partial V3 loopsequence, isolate HIV-1 SC (A)-(B)-(X)-Y-Asn-Asn-Thr-Thr-Arg- (SEQ IDNO:10) Ser-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Ala-Thr-Gly-Asp-Ile-Ile-Gly- Y-(X)-Z 4. Partial V3 loopsequence, isolate HIV-1 MN (A)-(B)-(X)-Y-Tyr-Asn-Lys-Arg-Lys- (SEQ IDNO:11) Arg-Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Lys-Asn-Ile-Ile-Gly- Y-(X)-Z 5. Partial V3 loopsequence, isolate HIV-1 RF (A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys- (SEQ IDNO:12) Ser-Ile-Thr-Lys-Gly-Pro-Gly-Arg-Val-Ile-Tyr-Ala-Thr-Gly-Gln-Ile-Ile-Gly- Y-(X)-Z 6. Partial V3 loopsequence, isolate HIV-1 mal (A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Arg- (SEQ IDNO:13) Gly-Ile-His-Phe-Gly-Pro-Gly-Gln-Ala-Leu-Tyr-Thr-Thr-Gly-Ile-Val-Gly-Y- (X)-Z 7. Partial V3 loop sequence,isolate HTLV-IIIB (A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys- (SEQ ID NO:14)Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile- Gly-Y-(X)-Z 8. Partial V3 loopsequence, isolate HIV-1 ELI (A)-(B)-(X)-Y-Gln-Asn-Thr-Arg-Gln- (SEQ IDNO:15) Arg-Thr-Pro-Ile-Gly-Leu-Gly-Gln-Ser-Leu-Tyr-Thr-Thr-Arg-Ser-Arg-Ser-Y- (X)-Z 9. Partial V3 loop sequence,isolate ANT70 (A)-(B)-(X)-Y-Gln-Ile-Asp-Ile-Gln- (SEQ ID NO:16)Glu-Met-Arg-Ile-Gly-Pro-Met-Ala-Trp- Tyr-Ser-Met-Gly-Ile-Gly-Gly-Y-(X)-Z10. Partial V3 loop sequence, Brazilian isolate, Peptide V3-368(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Arg- (SEQ ID NO:17)Gly-Ile-His-Met-Gly-Trp-Gly-Arg-Thr-Phe-Tyr-Ala-Thr-Gly-Glu-Ile-Ile-Gly- Y-(X)-Z 11. Carboxy-terminus, HIV-1gp120 (A)-(B)-(X)-Y-Arg-Asp-Asn-Trp-Arg- (SEQ ID NO:18)Ser-Glu-Leu-Tyr-Lys-Tyr-Lys-Val-Val-Lys-Ile-Glu-Pro-Leu-Gly-Val-Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln- Arg-Glu-Lys-Arg-Y-(X)-Z 2. Humanimmunodeficiency Virus type 2 Envelope Peptide a. gp41, isolate HIV-2rod (A)-(B)-(X)-Y-Ser-Trp-Gly-Cys-Ala- (SEQ ID NO:19)Phe-Arg-Gln-Val-Cys-Y-(X)-Z b. (A)-(B)-(X)-Y-Lys-Tyr-Leu-Gln-Asp- (SEQID NO:20) Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y-(X)-Z c. gp120, isolate HIV-2 NiHZ(A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Leu- (SEQ ID NO:21)Pro-Ile-Thr-Phe-Met-Ser-Gly-Phe-Lys-Phe-His-Ser-Gln-Pro-Val-Ile-Asn-Lys- Y-(X)-Z d. Partial V3 loopsequence, Peptide V3-GB12 (A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Val- (SEQ IDNO:22) Pro-Ile-Thr-Leu-Met-Ser-Gly-Leu-Val-Phe-His-Ser-Gln-Pro-Ile-Asn-Lys-Y- (X)-Z e. Partial V3 loop sequence,Peptide V3-239 (A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Leu- (SEQ ID NO:23)Pro-Val-Thr-Ile-Met-Ser-Gly-Leu-Val- Phe-His-Ser-Gln-Pro-Ile-Asn-Asp-Y-(X)-Z 3. Chimpanzee immunodeficiency Virus a. gp41(A)-(B)-(X)-Y-Leu-Trp-Gly-Cys-Ser- (SEQ ID NO:24)Gly-Lys-Ala-Val-Cys-Y-(X)-Z 4. Simian immunodeficiency Virus a.Transmembrane protein, isolate SIVagm (TY01)(A)-(B)-(X)-Y-Ser-Trp-Gly-Cys-Ala- (SEQ ID NO:25)Trp-Lys-Gln-Val-Cys-Y-(X)-Z b. Transmembrane protein, isolate SIVmnd(A)-(B)-(X)-Y-Gln-Trp-Gly-Cys-Ser- (SEQ ID NO:26)Trp-Ala-Gln-Val-Cys-Y-(X)-Z 5. HTLV-I and HTLV-II Virus Peptide I-gp46-3(A)-(B)-(X)-Y-Val-Leu-Tyr-Ser-Pro- (SEQ ID NO:27)Asn-Val-Ser-Val-Pro-Ser-Ser-Ser-Ser- Thr-Leu-Leu-Tyr-Pro-Ser-Leu-Ala-Y-(X)-Z Peptide I-gp46-5 (A)-(B)-(X)-Y-Tyr-Thr-Cys-Ile-Val- (SEQ ID NO:28)Cys-Ile-Asp-Arg-Ala-Ser-Leu-Ser-Thr- Trp-His-Val-Leu-Tyr-Ser-Pro-Y-(X)-ZPeptide I-gp46-4 (A)-(B)-(X)-Y-Asn-Ser-Leu-Ile-Leu- (SEQ ID NO:29)Pro-Pro-Phe-Ser-Leu-Ser-Pro-Val-Pro- Thr-Leu-Gly-Ser-Arg-Ser-Arg-Arg-Y-(X)-Z Peptide I-gp46-6 (A)-(B)-(X)-Y-Asp-Ala-Pro-Gly-Tyr- (SEQ ID NO:30)Asp-Pro-Ile-Trp-Phe-Leu-Asn-Thr-Glu-Pro-Ser-Gln-Leu-Pro-Pro-Thr-Ala-Pro-Pro-Leu-Leu-Pro-His-Ser-Asn-Leu-Asp- His-Ile-Leu-Glu-Y-(X)-Z PeptideI-p21-2 (A)-(B)-(X)-Y-Gln-Tyr-Ala-Ala-Gln- (SEQ ID NO:31)Asn-Arg-Arg-Gly-Leu-Asp-Leu-Leu-Phe-Trp-Glu-Gln-Gly-Gly-Leu-Cys-Lys-Ala- Leu-Gln-Glu-Gln-Cys-Arg-Phe-Pro-Y-(X)-Z Peptide I-p19 (A)-(B)-(X)-Y-Pro-Pro-Pro-Pro-Ser- (SEQ ID NO:32)Ser-Pro-Thr-His-Asp-Pro-Pro-Asp-Ser-Asp-Pro-Gln-Ile-Pro-Pro-Pro-Tyr-Val- Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu-Y-(X)-Z Peptide II-gp52-1 (A)-(B)-(X)-Y-Lys-Lys-Pro-Asn-Arg- (SEQ IDNO:33) Gln-Gly-Leu-Gly-Tyr-Tyr-Ser-Pro-Ser- Tyr-Asn-Asp-Pro-Y-(X)-ZPeptide II-gp52-2 (A)-(B)-(X)-Y-Asp-Ala-Pro-Gly-Tyr- (SEQ ID NO:34)Asp-Pro-Leu-Trp-Phe-Ile-Thr-Ser-Glu-Pro-Thr-Gln-Pro-Pro-Pro-Thr-Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu- His-Val-Leu-Thr-Y-(X)-Z PeptideII-gp52-3: (A)-(B)-(X)-Y-Tyr-Ser-Cys-Met-Val- (SEQ ID NO:35)Cys-Val-Asp-Arg-Ser-Ser-Leu-Ser-Ser-Trp-His-Val-Leu-Tyr-Thr-Pro-Asn-Ile-Ser-Ile-Pro-Gln-Gln-Thr-Ser-Ser-Arg- Thr-Ile-Leu-Phe-Pro-Ser-Y-(X)-ZPeptide II-p19 (A)-(B)-(X)-Y-Pro-Thr-Thr-Thr-Pro- (SEQ ID NO:36)Pro-Pro-Pro-Pro-Pro-Pro-Ser-Pro-Glu-Ala-His-Val-Pro-Pro-Pro-Tyr-Val-Glu- Pro-Thr-Thr-Thr-Gln-Cys-Phe-Y-(X)-Z

These above-mentioned biotinylated peptides were synthesized and foundto be specifically recognized by antisera from infected humans orprimates are considered particularly advantageous. All theseabove-mentioned peptides are new. The process of the invention enablesto increase the antigenicity of these HIV peptides, which can however bebound to a support, even when they are not biotinylated.

The HCV peptide sequences, which follow, have been found to bespecifically recognized by antisera from infected humans or primates andwhich are considered particularly advantageous. The non-biotinylatedamino acid sequences can be synthesized according to classical methods.

The peptides of interest are intended to mimic immunologically proteinsor domains of proteins encoded by HCV. Since sequence variability hasbeen observed for HCV, it may be desirable to vary one or more aminoacids so as to better mimic the epitopes of different strains. It shouldbe understood that the peptides described need not be identical to anyparticular HCV sequence as long as the subject compounds are capable ofproviding for immunological competition with at least one strain of HCV.

The peptides may therefore be subject to insertions, deletions andconservative as well as non-conservative amino acid substitutions wheresuch changes might provide for certain advantages in their use. Thepeptides will preferably be as short as possible while still maintainingall of the sensitivity of the larger sequence. In certain cases, it maybe desirable to join two or more peptides together into a singlestructure.

The formation of such a composite may involve covalent or non-covalentlinkages.

Of particular interest are biotinylated peptides of HCV into whichcysteine, thioglycollic acid, or other thiol-containing compounds havebeen incorporated into the peptide chain for the purpose of providingmercapto-groups, which can be used, for cyclization of the peptides.

The following peptides from the Core region of HCV were determined ascorresponding to immunologically important epitopes. 1. Peptide I orCore I (aa. 1-20) has the following amino acid sequence: (I)(A)-(B)-(X)-Y-Met-Ser-Thr-Ile-Pro- (SEQ ID NO:37)Lys-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn- Thr-Asn-Arg-Arg-Pro-Gln-Y-(X)-Z 2.Peptide II or Core 2 (aa. 7-26) has the amino acid sequence: (II)(A)-(B)-(X)-Y-Pro-Gln-Arg-Lys-Thr- (SEQ ID NO:38)Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln- Asp-Val-Lys-Phe-Pro-Gly-Y-(X)-Z Ofparticular interest is the oligopeptide IIA (aa. 8 to 18): (IIA)(A)-(B)-(X)-Y-Gln-Arg-Lys-Thr-Lys- (SEQ ID NO:39)Arg-Asn-Thr-Asn-Arg-Arg-Y-(X)-Z 3. Peptide III or Core 3 (aa 13-32) hasthe sequence: (III) (A)-(B)-(X)-Y-Arg-Asn-Thr-Asn-Arg- (SEQ ID NO:40)Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly- Gly-Gly-Gln-Ile-Val-Gly-Y-(X)-Z 4.Peptide IV or Core 7 (aa 37-56) has the sequences: (IV)(A)-(B)-(X)-Y-Leu-Pro-Arg-Arg-Gly- (SEQ ID NO:41)Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Arg- Lys-Thr-Ser-Glu-Arg-Ser-Y-(X)-Z Ofparticular interest is the oligopeptide. IVa or Core 6 (aa. 31 to 50):(IVa) (A)-(B)-(X)-Y-Val-Gly-Gly-Val-Tyr- (SEQ ID NO:42)Leu-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu- Gly-Val-Arg-Ala-Thr-Arg-Y-(X)-Z 5.Peptide V or Core 9 (aa 49-68) has the sequence: (V)(A)-(B)-(X)-Y-Thr-Arg-Lys-Thr-Ser- (SEQ ID NO:43)Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg- Gln-Pro-Ile-Pro-Lys-Val-Y-(X)-Z Ofparticular interest is the oligopeptide Va (aa. 55 to 74): (Va)(A)-(B)-(X)-Y-Arg-Ser-Gln-Pro-Arg- (SEQ ID NO:44)Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val- Arg-Arg-Pro-Glu-Gly-Arg-Y-(X)-Z 6.Peptide VI or Core 11 (aa 61-80) has the following sequence: (VI)(A)-(B)-(X)-Y-Arg-Arg-Gln-Pro-Ile- (SEQ ID NO:45)Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg- Thr-Trp-Ala-Gln-Pro-Gly-Y-(X)-Z 7.Peptide VII (aa 73-92) or core 13 has the sequence: (VII)(A)-(B)-(X)-Y-Gly-Arg-Thr-Trp-Ala- (SEQ ID NO:46)Gln-Pro-Gly-Tyr-Pro-Trp-Pro-Leu-Tyr- Gly-Asn-Glu-Gly-Cys-Gly-Y-(X)-Z 8.Peptide Core 123 (aa. 1-32) (A)-(B)-(X)-Y-Met-Ser-Thr-Ile-Pro- (SEQ IDNO:47) Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro- Gly-Gly-Gly-Gln-Ile-Val-Gly-Y-(X)-Z9. Peptide Core 7910 (aa. 37-80): (A)-(B)-(X)-Y-Gly-Gly-Val-Tyr-Leu-(SEQ ID NO:48) Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Arg- Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Y-(X)-Z

The following peptides from the NS4 region of HCV were found tocorrespond to immunologically important epitopes. Peptide VIII or NS4-1or HCV1 (aa 1688-1707) has the sequence: (VIII)(A)-(B)-(X)-Y-Leu-Ser-Gly-Lys-Pro- (SEQ ID NO:49)Ala-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu- Tyr-Arg-Glu-Phe-Asp-Glu-Y-(X)-ZPeptide IX or HCV2 (aa 1694-1713) has the sequence: (IX)(A)-(B)-(X)-Y-Ile-Ile-Pro-Asp-Arg- (SEQ ID NO:50)Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu- Met-Glu-Glu-Cys-Ser-Gln-Y-(X)-ZPeptide HCV3 (A)-(B)-(X)-Y-Val-Leu-Tyr-Arg-Glu- (SEQ ID NO:51)Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln- His-Leu-Pro-Tyr-Ile-Glu-Y-(X)-ZPeptide X or HCV4 (aa 1706-1725) has the sequence: (X)(A)-(B)-(X)-Y-Asp-Glu-Met-Glu-Glu- (SEQ ID NO:52)Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu- Gln-Gly-Met-Met-Leu-Ala-Y-(X)-Z 11.Peptide XI or NS4-5 or HCV5 (aa 1712-1731) has the sequence: (XI)(A)-(B)-(X)-Y-Ser-Gln-His-Leu-Pro- (SEQ ID NO:53)Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala- Glu-Gln-Phe-Lys-Gln-Eys-Y-(X)-Z 12.Peptide XII or HCV6 (aa 1718-1737) has the sequence: (XII)(A)-(B)-(X)-Y-Ile-Glu-Gln-Gly-Met- (SEQ ID NO:54)Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys- Ala-Leu-Gly-Leu-Leu-Gln-Y-(X)-Z 13.Peptide XIII or NS4-7 or HCV7 (aa 1724-1743) has the sequence: (XIII)(A)-(B)-(X)-Y-Leu-Ala-Glu-Gln-Phe- (SEQ ID NO:55)Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln- Thr-Ala-Ser-Arg-Gln-Ala-Y-(X)-Z 14.Peptide XIV or HCV8 (aa 1730-1749) has the sequence: (XIV)(A)-(B)-(X)-Y-Gln-Lys-Ala-Leu-Gly- (SEQ ID NO:56)Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala- Glu-Val-Ile-Ala-Pro-Ala-Y-(X)-Z 15.Peptide NS4-27 or HCV9 (aa. 1712-1743):(A)-(B)-(X)-Y-Ser-Gln-His-Leu-Pro- (SEQ ID NO:57)Tyr-Ile-Glu-Gln-Glu-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu- Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Y-(X)-Z 16. Peptide NS4e: (A)-(B)-(X)-Y-Gly-Glu-Gly-Ala-Val- (SEQ IDNO:58) Gln-Trp-Met-Asn-Arg-Leu-Ile-Ala-Phe-Ala-Ser-Arg-Gly-Asn-His-Y-(X)-Z

The following peptides of the NS5 region of HCV were found to correspondto immunologically important epitopes. Peptide XV or NS5-25 (aa2263-2282) has the sequence: (XV) (A)-(B)-(X)-Y-Glu-Asp-Glu-Arg-Glu-(SEQ ID NO:59) Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Y-(X)-Z Peptide XVI or NS5-27 (aa 2275-2294) hasthe sequence: (XVI) (A)-(B)-(X)-Y-Leu-Arg-Lys-Ser-Arg- (SEQ ID NO:60)Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val′Trp- Ala-Arg-Pro-Asp-Tyr-Asn-Y-(X)-ZPeptide XVII or NS5-29 (aa 2287-2306) has the sequence: (XVII)(A)-(B)-(X)-Y-Val-Trp-Ala-Arg-Pro- (SEQ ID NO:61)Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr- Trp-Lys-Lys-Pro-Asp-Tyr-Y-(X)-ZPeptide XVIII or NS5-31 (aa 2299-2318) has the sequence: (XVIII)(A)-(B)-(X)-Y-Glu-Thr-Trp-Lys-Lys- (SEQ ID NO:62)Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His- Gly-Cys-Pro-Leu-Pro-Pro-Y-(X)-ZPeptide XIX or NS5-33 (aa 2311-2330) has the sequence: (XIX)(A)-(B)-(X)-Y-Val-His-Gly-Cys-Pro- (SEQ ID NO:63)Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val- Pro-Pro-Pro-Arg-Lys-Lys-Y-(X)-ZPeptide NS5-2527 (aa. 2263 to 2294): (A)-(B)-(X)-Y-Glu-Asp-Glu-Arg-Glu-(SEQ ID NO:64) Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-Lys-Ser-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro- Asp-Tyr-Asp-Tyr-Asn-Y-(X)-Z

The following peptides from the N-terminal region of the E2/NS1 regionof HCV were found to correspond to immunologically important epitopes.Peptide XXa (aa. 383-416) (A)-(B)-(X)-Y-Gly-Glu-Thr-Tyr-Thr- (SEQ IDNO:65) Ser-Gly-Gly-Ala-Ala-Ser-His-Thr-Thr-Ser-Thr-Leu-Ala-Ser-Leu-Phe-Ser-Pro-Gly-Ala-Ser-Gln-Arg-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXa-1 (aa.383-404) (A)-(B)-(X)-Y-Gly-Glu-Thr-Tyr-Thr- (SEQ ID NO:66)Ser-Gly-Gly-Ala-Ala-Ser-His-Thr-Thr- Ser-Thr-Leu-Ala-Ser-Leu-Phe-Ser-Y-(X)-Z Peptide XXa-2 (aa. 393-416) (A)-(B)-(X)-Y-Ser-His-Thr-Thr-Ser-(SEQ ID NO:67) Thr-Leu-Ala-Ser-Leu-Phe-Ser-Pro-Gly-Ala-Ser-Gln-Arg-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXb (aa.383-416) (A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val- (SEQ ID NO:68)Ser-Gly-Gly-Ala-Ala-Ala-Ser-Asp-Thr-Arg-Gly-Leu-Val-Ser-Leu-Phe-Ser-Pro-Gly-Ser-Ala-Gln-Lys-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXb-1 (aa.383-404) (A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val- (SEQ ID NO:69)Ser-Gly-Gly-Ala-Ala-Ala-Ser-Asp-Thr- Arg-Gly-Leu-Val-Ser-Leu-Phe-Ser-Y-(X)-Z Peptide XXb-2 (aa. 393-416) (A)-(B)-(X)-Y-Ala-Ser-Asp-Thr-Arg-(SEQ ID NO:70) Gly-Leu-Val-Ser-Leu-Phe-Ser-Pro-Gly-Ser-Ala-Gln-Lys-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXc (aa.383-416) (A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val- (SEQ ID NO:71)Thr-Gly-Gly-Val-Gln-Gly-His-Val-Thr-Cys-Thr-Leu-Thr-Ser-Leu-Phe-Arg-Pro-Gly-Ala-Ser-Gln-Lys-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXc-1 (aa.383-404) (A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val- (SEQ ID NO:72)Thr-Gly-Gly-Val-Gln-Gly-His-Val-Thr- Cys-Thr-Leu-Thr-Ser-Leu-Phe-Arg-Y-(X)-Z Peptide XXc-2 (aa. 393-416) (A)-(B)-(X)-Y-Gly-His-Val-Thr-Cys-(SEQ ID NO:73) Thr-Leu-Thr-Ser-Leu-Phe-Arg-Pro-Gly-Ala-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXd (aa.383-416) (A)-(B)-(X)-Y-Gly-His-Thr-His-Val- (SEQ ID NO:74)Thr-Gly-Gly-Arg-Val-Ala-Ser-Ser-Thr-Gln-Ser-Leu-Val-Ser-Trp-Leu-Ser-Gln-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXd-1 (aa.383-404) (A)-(B)-(X)-Y-Gly-His-Thr-His-Val- (SEQ ID NO:75)Thr-Gly-Gly-Arg-Val-Ala-Ser-Ser-Thr- Gln-Ser-Leu-Val-Ser-Trp-Leu-Ser-Y-(X)-Z Peptide XXd-2 (aa. 393-416) (A)-(B)-(X)-Y-Ala-Ser-Ser-Thr-Gln-(SEQ ID NO:76) Ser-Leu-Val-Ser-Trp-Leu-Ser-Gln-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXe (aa.383-416) (A)-(B)-(X)-Y-Gly-Asp-Thr-His-Val- (SEQ ID NO:77)Thr-Gly-Gly-Ala-Gln-Ala-Lys-Thr-Thr-Asn-Arg-Leu-Val-Ser-Met-Phe-Ala-Ser-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXe-1 (aa.383-404) (A)-(B)-(X)-Y-Gly-Asp-Thr-His-Val- (SEQ ID NO:78)Thr-Gly-Gly-Ala-Gln,Ala-Lys-Thr-Thr- Asn-Arg-Leu-Val-Ser-Met-Phe-Ala-Y-(X)-Z Peptide XXe-2 (aa. 393-416) (A)-(B)-(X)-Y-Ala-Lys-Thr-Thr-Asn-(SEQ ID NO:79) Arg-Leu-Val-Ser-Met-Phe-Ala-Ser-Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Ile-Asn- Thr-Y-(X)-Z Peptide XXf (aa.383-416) (A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr″ (SEQ ID NO:80)Ser-Gly-Gly-Asn-Ala-Gly-His-Thr-Met-Thr-Gly-Ile-Val-Arg-Phe-Phe-Ala-Pro-Gly-Pro-Lys-Gln-Asn-Val-His-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXf-1 (aa.383-404) (A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr- (SEQ ID NO:81)Ser-Gly-Gly-Asn-Ala-Gly-His-Thr-Met- Thr-Gly-Ile-Val-Arg-Phe-Phe-Ala-Y-(X)-Z Peptide XXf-2 (aa. 393-416) (A)-(B)-(X)-Y-Gly-His-Thr-Met-Thr-(SEQ ID NO:82) Gly-Ile-Val-Arg-Phe-Phe-Ala-Pro-Gly-Pro-Lys-Gln-Asn-Val-His-Leu-Ile-Asn- Thr-Y-(X)-Z Peptide XXg (aa.383-416) (A)-(B)-(X)-Y-Ala-Glu-Thr-Ile-Val- (SEQ ID NO:83)Ser-Gly-Gly-Gln-Ala-Ala-Arg-Ala-Met-Ser-Gly-Leu-Val-Ser-Leu-Phe-Thr-Pro-Gly-Ala-Lys-Gln-Asn-Ile-Gln-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXg-1 (aa.383-404) (A)-(B)-(X)-Y-Ala-Glu-Thr-Ile-Val- (SEQ ID NO:84)Ser-Gly-Gly-Gln-Ala-Ala-Arg-Ala-Met- Ser-Gly-Leu-Val-Ser-Leu-Phe-Thr-Y-(X)-Z Peptide XXg-2 (aa. 393-416) (A)-(B)-(X)-Y-Ala-Arg-Ala-Met-Ser-(SEQ ID NO:85) Gly-Leu-Val-Ser-Leu-Phe-Thr-Pro-Gly-Ala-Lys-Gln-Asn-Ile-Gln-Leu-Ile-Asn- Thr-Y-(X)-Z Peptide XXh (aa.383-416) (A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr- (SEQ ID NO:86)Thr-Gly-Gly-Ser-Thr-Ala-Arg-Thr-Thr-Gln-Gly-Leu-Val-Ser-Leu-Phe-Ser-Arg-Gly-Ala-Lys-Gln-Asp-Ile-Gln-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXh-1 (aa.383-404) (A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr- (SEQ ID NO:87)Thr-Gly-Gly-Ser-Thr-Ala-Arg-Thr-Thr- Gln-Gly-Leu-Val-Ser-Leu-Phe-Ser-Y-(X)-Z Peptide XXh-2 (aa. 393-416) (A)-(B)-(X)-Y-Ala-Arg-Thr-Thr-Gln-(SEQ ID NO:88) Gly-Leu-Val-Ser-Leu-Phe-Ser-Arg-Gly-Ala-Lys-Gln-Asp-Ile-Gln-Leu-Ile-Asn- Thr-Y-(X)-Z

The above-mentioned sequences correspond to epitopes localized on theHCV type-1 isolate HCV-1 (Choo et al. Proc; Natl. Acad. Sci. 88,2451-2455, 1991) and HC-J1 (Okamoto et al., Jap. J. Exp. Med. 60,167-177, 1990) sequence. It is, however, to be understood that alsopeptides from other type-1 HCV isolate sequences which correspond to theabove-mentioned immunologically important regions may also be comprisedin the composition according to the invention. An example of variant HCVsequences also falling within the present invention may be derived fromthe HCV-J isolate (Kato et al., Proc. Natl. Acad. Sci. 87, 9524-9528).

The following peptides derived from the same regions as the above-citedpeptide regions from the type 2 HCV sequences Peptide XX/2(A)-(B)-(X)-Y-Ala-Gln-Thr-His-Thr- (SEQ ID NO:89)Val-Gly-Gly-Ser-Thr-Ala-His-Asn-Ala-Arg-Thr-Leu-Thr-Gly-Met-Phe-Ser-Leu-Gly-Ala-Arg-Gln-Lys-Ile-Gln-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XX/2-1(A)-(B)-(X)-Y-Ala-Gln-Thr-His-Thr- (SEQ ID NO:90)Val-Gly-Gly-Ser-Thr-Ala-His-Asn-Ala- Arg-Thr-Leu-Thr-Gly-Met-Phe-Ser-Y-(X)-Z Peptide XX/2-2 (A)-(B)-(X)-Y-Ala-His-Asn-Ala-Arg- (SEQ ID NO:91)Thr-Leu-Thr-Gly-Met-Phe-Ser-Leu-Gly-Ala-Arg-Gln-Lys-Ile-Gln-Leu-Ile-Asn- Thr-Y-(X)-Z Peptide VIII-2 or NS4-1(2) (A)-(B)-(X)-Y-Val-Asn-Gln-Arg-Ala- (SEQ ID NO:92)Val-Val-Ala-Pro-Asp-Lys-Glu-Val-Leu- Tyr-Glu-Ala-Phe-Asp-Glu-Y-(X)-ZPeptide IX-2 (A)-(B)-(X)-Y-Val-Ala-Pro-Asp-Lys- (SEQ ID NO:93)Glu-Val-Leu-Tyr-Glu-Ala-Phe-Asp-Glu- Met-Glu-Glu-Cys-Ala-Ser-Y-(X)-ZPeptide X-2 (A)-(B)-(X)-Y-Asp-Glu-Met-Glu-Glu- (SEQ ID NO:94)Cys-Ala-Ser-Arg-Ala-Ala-Leu-Ile-Glu- Glu-Gly-Gln-Arg-Ile-Ala-Y-(X)-ZPeptide XI-2 or NS4-5 (2) (A)-(B)-(X)-Y-Ala-Ser-Arg-Ala-Ala- (SEQ IDNO:95) Leu-Ile-Glu-Glu-Gly-Gln-Arg-Ile-Ala-Glu-Met-Leu-Lys-Ser-Lys-Y-(X)-Z Peptide XII-2(A)-(B)-(X)-Y-Ile-Glu-Glu-Gly-Gln- (SEQ ID NO:96)Arg-Ile-Ala-Glu-Met-Leu-Lys-Ser-Lys- Ile-Gln-Gly-Leu-Leu-Gln-Y-(X)-ZPeptide XIII-2 or NS4-7 (2) (A)-(B)-(X)-Y-Ile-Ala-Glu-Met-Leu- (SEQ IDNO:97) Lys-Ser-Lys-Ile-Gln-Gly-Leu-Leu-Gln-Gln-Ala-Ser-Lys-Gln-Ala-Y-(X)-Z Peptide XIV-2(A)-(B)-(X)-Y-Ser-Lys-Ile-Gln-Gly- (SEQ ID NO:98)Leu-Leu-Gln-Gln-Ala-Ser-Lys-Gln-Ala- Gln-Asp-Ile-Gln-Pro-Ala-Y-(X)-ZPeptide XV-2 (A)-(B)-(X)-Y-Arg-Ser-Asp-Leu-Glu- (SEQ ID NO:99)Pro-Ser-Ile-Pro-Ser-Glu-Tyr-Met-Leu- Pro-Lys-Lys-Arg-Phe-Pro-(X)-Y-ZPeptide XVI-2 (A)-(B)-(X)-Y-Met-Leu-Pro-Lys-Lys- (SEQ ID NO:100)Arg-Phe-Pro-Pro-Ala-Leu-Pro-Ala-Trp- Ala-Arg-Pro-Asp-Tyr-Asn-Y-(X)-ZPeptide XVII-2 (A)-(B)-(X)-Y-Ala-Trp-Ala-Arg-Pro- (SEQ ID NO:101)Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Ser- Trp-Lys-Arg-Pro-Asp-Tyr-Y-(X)-ZPeptide XVIII-2 (A)-(B)-(X)-Y-Glu-Ser-Trp-Lys-Arg- (SEQ ID NO:102)Pro-Asp-Tyr-Gln-Pro-Ala-Thr-Val-Ala- Gly-Cys-Ala-Leu-Pro-Pro-Y-(X)-ZPeptide XIX-2 (A)-(B)-(X)-Y-Val-Ala-Gly-Cys-Ala- (SEQ ID NO:103)Leu-Pro-Pro-Pro-Lys-Lys-Thr-Pro-Thr- Pro-Pro-Pro-Arg-Arg-Arg-Y-(X)-Z

The above-mentioned sequences correspond to epitopes localized on theHCV type-2 isolate HC-J6 sequence (Okamoto et al., J. Gen. Virology 72,2697-2704, 1991). It is however, to be understood that also peptidesfrom other type-2 HCV isolate sequences which correspond to theabove-mentioned immunologically important regions may also be comprisedin the composition according to the invention. Examples of variantsequences also falling within the present invention may be derived fromHCV isolate HC-J8 (Okamato et al., Virology 188, 331-341, 1992).

The following peptides from the NS4 region of HCV type 3 are alsopreferred peptides according to the; present invention: Peptide NS4-1(3) (A)-(B)-(X)-Y-Leu-Gly-Gly-Lys-Pro- (SEQ ID NO:107)Ala-Ile-Val-Pro-Asp-Lys-Glu-Val-leu- Tyr-Gln-Gln-Tyr-Asp-Glu-Y-(X)-ZPeptide NS4-5 (3) (A)-(B)-(X)-Y-Ser-Gln-Ala-Ala-Pro- (SEQ ID NO:108)Tyr-Ile-Glu-Gln-Ala-Gln-Val-Ile-Ala- His-Gln-Phe-Lys-Glu-Lys-Y-(X)-ZPeptide NS4-7 (3) (A)-(B)-(X)-Y-Ile-Ala-His-Gln-His- (SEQ ID NO:109)Gln-Phe-Lys-Glu-Lys-Val-Leu-Gly-Leu- Leu-Gln-Arg-Ala-Thr-Gln-Gln-Gln-Y-(X)-Z

It is to be understood that also other peptides corresponding to HCVtype-3 isolate sequences which YAC) 13/18054 PCF/EP93/00517 correspondto immunologically important regions as determined for HCV type-1 andtype-2 may also be comprised in the composition according to theinvention.

The composition according to the present invention may also comprisehybrid HCV peptide sequences consisting of combinations of the coreepitopes of the HCV core (table 9) HCV NS4 (table 10) or the HCV N5(table 11) region separated by Gly and/or Ser residues, andpreferentially the following hybrid HCV sequences: Epi-152(A)-(B)-(X)-Y-Ile-Pro-Asp-Arg-Glu- (SEQ ID NO:104)Val-Leu-Tyr-Arg-Gly-Gly-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-Gly-Gly-Arg- Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Y-(X)-Z Epi-33B3A (A)-(B)-(X)-Y-Trp-Ala-Arg-Pro-Asp- (SEQ ID NO:105)Tyr-Asn-Pro-Pro-Gly-Gly-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Gly-Ser-Gly- Val-Tyr-Leu-Leu-Pro-Arg-Arg-Gly-Y-(X)-Z Epi-4B2A6 (A)-(B)-(X)-Y-Arg-Gly-Arg-Arg-Gln- (SEQ ID NO:106)Pro-Ile-Pro-Lys-Gly-Gly-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Ser-Gly-Pro- Val-Val-His-Gly-Cys-Pro-Leu-Pro-Y-(X)-Z

The composition according to the present invention may also comprise socalled biotinylated mixotope sequences consisting of peptides containingat each position all the amino acids found in the naturally occurringisolates, with said peptides being derived from any of theabove-mentioned immunologically important regions (see FIG. 14).

(2) A preferred mixture of biotinylated peptides for detecting and/orimmunizing against Hepatitis C Virus, Human Immunodeficiency Virus type1 and human Immunodeficiency Virus type 2 consists of:

-   A. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII, 1a.3, 1a.4, 1a.b,    1b.1a, 2b, 2d,-   B. II, III, IVa, Va, IX, IX-2, XI, XI-2, XIII, XIII-2, XV, XV-2,    XVI, XVI-2, XVIII, XVIII-2, 1a.3, 1a.4, 1a.b, 1b.1a, 2b, 2d.

(3) A preferred mixture of biotinylated peptides for detecting and/orimmunizing against Human Immunodef iciency Virus types 1 and 2 and HumanLymphotropic Virus types I and II consists of:

-   1a.3, 1a.4, 1b.1, 2b, 2c, 2d, I-gp46-3, I-gp46-4, I-gp46-5,    I-gp46-6, II-gp52-2, II-gp52-3, I-p21-2, I-p19, II-p19.

(4) Another preferred mixture of biotinylated peptides for detectingand/or immunizing against Hepatitis C Virus, Human ImmunodeficiencyVirus types 1 and 2 and Human Lymphotropic virus types I and II consistsof:

-   1a.3, 1a.4, 1a.6, 1b.1a, 2d, II, III, IVa, Va, IX, XI, XIII, XV,    XVI, XVIII, XXa-2, XXc-2, XXg-2,-XXh-2, I-gp46-3, I-gp46-4,    I-gp46-5, I-gp46-6, II-gp52-3, I-p21-2, I-P19, II-P19.

(5) The present invention relates also to compositions of biotinylatedpeptides which ate considered particularly advantageous, for diagnosticas well as immunogenic purpose for Hepatitis C Virus, and whichadvantageously comprise the following mixtures:

-   A. I, III, IVa, Va,-   B. II, III, IVa, Va,-   C. IX, XI, XIII,-   D. XV, XVI, XVIII, XIX,-   E. XXc-2, XXa-1, XXa-2, XXh-1, XXh-2, XXg-2, XX/2-2,-   F. IX-2, XI-2, XIII-2,-   G. XV-2, XVI-2, XVIII-2, XIX-2,-   H. IX, IX-2,. XI, XI-2, XIII,XIII-2,-   I. XV, XV-2, XVI, XVI-2, XVIII, XVIII-2, XIX, XIX-2,-   J. II, III, IVa, Va, IX, IX-2, XI, XI-2, XIII, XIII-2, XV, XV-2,    XVI, XVI-2, XVIII,XVIII-2,-   K. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII,-   L. II, III,IV, V, IX, XI, XIII, XV, XVI, XVIII,-   M. II, III, IVa, Va, IX, XI, XIII,XV, XVI, XVIII, XXa-2, XXc-2,    XXg-2, XXh-2.

(6) The present invention relates also to compositions of biotinylatedpeptides which are considered particularly advantageous, for diagnosticas well as immunogenic purposes for Human Immunodeficiency virus, andwhich are advantageously selected from the following mixtures:

For type 1:

-   -   A. 1a.3, 1a.4, 1a.5, 1a.b    -   B. 1a.3, 1a.4, 1b.1, 1b.3, 1b.6, 1b.10,    -   C. 1b.1,1b.2, 1b.3, 1b.4, 1b.5, 1b.6, 1b.7, 1b.8, 1b.9, 1b.10    -   D. 1b.1, 1b.2, 1b.3,1b.4, 1b.6, 1b.10,    -   E. 1a.3, 1a.4, 1a.5, 1a.b, 1b.1a.        For type 2:    -   A. 2b, 2c, 2d, 2e.        For types 1 and 2:    -   A. 1a.3, 1a.4, 1b.1, 2b, 2c, 2d,    -   B. 1a.3, 1a.4, 1b.1a, 2b, 2d.

(7) The present invention relates also to compositions comprisingbiotinylated peptides, which are considered particularly advantageous,for diagnostic as well as immunogenic purposes for Human T-cellLymphotropic virus and are advantageously, selected from the followingmixtures:

For Human T-Lymphotropic virus type I:

-   -   Peptides I-gp46-3, I-gp46-4, I-gp46-5, I-gp46-6, I-p21-2, I-p19

For Human T-Lymphotropic virus type II

-   -   Peptides II-gp52-1, II-gp52-2, II-gp52-3, I-gp46-4, II-p19,        I-p21-2.

For Human lymphotropic virus types I and II:

-   -   Peptides I-qp46-3, I-qp46-4, I-gp46-5, I-gp46-6, II-gp52-1,        IIgp52-2, II-gp52-3, I-p21-2, I-P19, II-P19.

The synthesis of the peptides may be achieved in solution or on a solidsupport. Synthesis protocols generally employ t-butyloxycarbonyl- or9-fluorenylmethoxycarbonyl-protected activated amino acids.

The procedures for carrying out the synthesis, the amino acid activationtechniques, the types of side-chain production, and the cleavageprocedures used are amply described in, for example, Stewart and Young,Solid Phase Peptide Synthesis, 2nd Edition, Pierce; Chemical Company,1984; and Atherton and Sheppard, Solid Phase Peptide Synthesis, IRLPress, 1989.

(8) The present invention also relates to a process for in vitrodetermination of antibodies using the above defined biotinylatedpeptides, wherein said biotinylated peptides are preferably, in the formof streptavidin-biotinylated peptide complexes or avidin-biotinylatedpeptide complexes.

In the complex of streptavidin-biotinylated peptides oravidin-biotinylated peptides, the peptides may be biotinylated eitherN-terminally, C-terminally or internally.

This approach for the determination of antibodies is not limited withrespect to peptide length and avoids the difficulties inherent incoating peptides directly onto the solid phase for immunologicalevaluation.

The use of biotinylated peptides, in the process of the invention, makesthe anchorage of peptides to a solid support such that it leaves theiressential amino acids free to be recognized by antibodies.

The expression anchoring peptide to a solid support means the attachmentof the peptide to a support via covalent bonds or non-covalentinteractions such that the peptide becomes immobilized.

The solid support can be nitrocellulose, polystyrene, nylon or any othernatural or synthetic polymer.

The expression “their essential amino acids are left free to berecognized by antibodies” means that amino acid side chains of thepeptide proper are neither chemically modified in any way nor involvedin the interaction between the peptide and the solid phase.

The use of biotinylated peptides in the process of the invention enablessaid biotinylated peptides to be free to assume a wide range ofconformations, among which at least one is appropriate for the bindingof antibodies to said biotinylated peptides.

Any biotinylated peptide can be selected to be used in the process ofthe invention. However, some of them are able to be anchored on solidsupport and to react with antibodies specifically recognizing theepitope-within this peptide even without being biotinylated and withoutbeing involved in a complex of avidin of streptavidin. In this case, theuse of biotinylated peptides results in an apparent increase of theantigenicity of peptides with respect to the antigenicity observed whenthe peptides are not biotinylated. The expression “apparent” is meant toindicate an observed change obtained under similar test conditionswithout regard to the absolute cause of the observed change.

By “antigenicity” is meant the property of a peptide to be bound by anantibody.

By “increase of antigenicity” is meant that a positive signal isobtained for a dilution which is at least two times the dilution of thenon-biotinylated peptides. Said positive signal is of the same magnitudeas the one obtained for non-biotinylated peptides.

In other words, obtaining a positive signal can be obtained for asmaller amount of biotinylated peptide, compared to the amount ofnon-biotinylated peptide.

The present invention also illustrated a process for the identificationof epitopes in a protein sequence comprises the following steps:

-   -   the preparation of peptides corresponding to portions of the        amino acid sequence of the protein or polypeptide to be        analyzed, said peptides being either contiguous, or preferably        overlapping each other, the amount of overlapping being at least        3 amino acids, and preferably about 6 to about 12, the length of        the peptides being at least about 5 amino acids and no more than        about 50, preferably no more than about 40 amino acids, and more        preferably from 9 to about 30 amino acids, with said peptides        being characterized in that they are biotinylated;    -   binding the peptides to a solid phase through the interaction        between the biotinyl group and streptavidin or avidin and        measuring antibody binding to the individual peptides using        classical methods.

(9) The present invention also relates to a process for the in vitrodetermination of antibodies to HIV or diagnosis of HIV infection byusing a peptide composition as defined above in an immunoassayprocedure, wherein the biotinylated peptides used are in the form ofcomplexes of streptavidin-biotinylated or of: avidin-biotinylatedpeptides.

(10) The present invention relates also to a process for the in vitrodetermination of antibodies to HCV or diagnosis of HCV infection byusing a peptide composition as defined above in an immunoassayprocedure, wherein the biotinylated peptides used are in the form ofcomplexes of streptavidin-biotinylated or of: avidin-biotinylatedpeptides.

(11) The present invention relates also to a process for the in vitrodetermination of antibodies to HTLV I or II or diagnosis of HTLV I or IIinfection by using a peptide composition as defined above in anirrmunoassay procedure, wherein the biotinylated peptides used are inthe form of complexes of streptavidin-biotinylated or ofavidin-biotinylated peptides.

A preferred method for carrying out the in vitro determination ofantibodies is by means of an enzyme-linked immunosorbant assay (ELISA).This assay employs a solid phase which is generally a polystyrenemicrotiter plate or bead. The solid phase may, however, be any materialwhich is capable of binding a protein, either chemically via a covalentlinkage or by passive adsorption. In this regard, nylon-based membranesare also considered to be particularly advantageous. The solid phase iscoated with streptavidin or avidin and after a suitable period, excessunbound protein is removed by washing. Any unoccupied binding sites onthe solid phase are then blocked with an irrelevant protein such asbovine serum albumin or casein.

A solution containing the mixture or selection of biotinylated peptidesis subsequently brought into contact with the streptavidin- oravidin-coated surface and allowed to bind. Unbound peptide is removed bywashing. Alternatively biotinylated peptides are allowed to formcomplexes with either avidin or streptavidin. The resulting complexesare used to coat the solid phase. After a suitable incubation period,unbound complex is removed by washing. An appropriate dilution of anantiserum or other body fluid is brought into contact with the solidphase to which the peptide is bound. The incubation is carried out for atime necessary to allow the binding reaction to occur. Subsequently,unbound components are removed by washing the solid phase. The detectionof immune complexes is achieved by using heterologous antibodies whichspecifically bind to the antibodies present in the test serum and whichhave been conjugated with an enzyme, preferably but not limited toeither horseradish peroxidase, alkaline phosphatase, or β-galactosidase,which is capable of converting a colorless or nearly colorless substrateor co-substrate into a highly colored product or a product capable offorming a colored complex with a chromogen which can be detectedvisually or measured spectrophotometrically.

Other detection systems known in the art may however be employed andinclude those in which the amount of product formed is measuredelectrochemically or luminometrically. The detection system may alsoemploy radioactively labelled antibodies, in which case the amount ofimmune complex is quantified by scintillation counting or counting. Inprinciple, any type of immunological test for the detection ofantibodies may be used, as long as the test makes use of the complexbetween either streptavidin or avidin and (a) biotinylated peptide(s)synthesized as described.

Also included are competition assays in which streptavidin- oravidin-biotinylated peptide complexes in solution aria permitted tocompete with the solid phase-bound antigen for antibody binding orassays in which free peptide in solution is permitted to compete withsolid phase-bound streptavidin or avidin: biotinylated peptidecomplexes. By way of example, the many types of immunological assays forthe detection and quantitation of antibodies and antigen are discussedin detail (Tijssen, P., Practice and Theory of Enzyme Immunoassays,Elsevier Press, Amsterdam, Oxford, N.Y., 1985).

The immunological assays may be restricted to single biotinylatedpeptides. Preferably, however, a mixture of biotinylated peptides isused which includes more than one epitope derived from the infectiousagent(s) to which the presence of specific antibodies is to be measured.

Another preferred method for carrying out the in vitro determination ofantibody detection is the line immunoassay (LIA).

This method of antibody detection consists essentially of the followingsteps:

-   -   the antigens, in the form of biotinylated peptide:    -    streptavidin or avidin complexes, to be tested or used are        applied as parallel lines onto a membrane which is capable of        binding, covalently or non-covalently, the antigen to be tested,    -   unoccupied binding sites on the membrane are blocked with an        irrelevant protein such as casein or bovine serum albumin,    -   the membrane is cut into strips in a direction perpendicular to        the direction in which the antigen (biotinylated peptide) lines        are applied,    -   an appropriate dilution of an antiserum or other body fluid        (containing antibodies to be detected) is brought into contact        with a strip to which the antigens are bound and allowed to        incubate for a period of time sufficient to permit the binding        reaction to occur,    -   unbound components are removed by washing the strip,    -   the detection of immune complexes is achieved by incubating the        strip with heterologous antibodies which specifically bind to        the antibodies in the test serum and which have been conjugated        to an enzyme such as horseradish peroxidase,    -   the incubation is carried out for a period sufficient to allow        binding to occur,    -   the presence of bound conjugate is detected by addition of the        required substrate or co-substrates which are converted to a        colored product by the action of the enzyme,    -   the reactions are detected visually or may be quantified by        densitometry.

(12) As demonstrated in the Examples section the present inventionrelates also the use of a peptide composition as defined above, forimmunization against HIV, and/or HCV, and/or HTLV I or II infection.

(13) The present invention also relates to a method for preparing thebiotinylated peptides used in the invention involves the use ofN-α-Fmoc-X (N-y-biotin) or N-α-Fmoc-X (N-y-biotin) derivative, wherein Xrepresents

where n is at least 1 but less than 10 and is preferably between 2 and6, one amino group being attached to the Cα atom while the other beingattached to carbon Cy, which is the most distal carbon in the sidechain; or their esters obtained with alcohol ROH and more particularlypentafluorophenyl ester;

-   -   y representing position y with respect to the carbon atom        carrying the COOH group in the radical.

This biotin derivative will be called intermediary product, and theabove-defined intermediary products are new compounds determinedaccording to the process of the invention.

(14) In an advantageous method for preparing the compounds of theinvention, the intermediary product can be represented by one of thefollowing formula:N-α-Fmoc-(N-y-biotin) is N-α-Fmoc-lysine (ε-biotin) orN-α-Fmoc-ornithine (N-δ-biotin)

(15) The N-terminal biotinylated peptides can be prepared according tothe method which comprises the following steps:

-   -   addition of the successive amino acids duly protected onto the        resin to give:        Fmoc-AA_(n) . . . AA₁-resin,    -   deprotection of the NH₂-terminal for instance by means of        piperidine,    -   addition of the intermediary product:    -    through its COOH onto the NH₂-terminal to obtain:    -   deprotection of the NH₂-terminal group of the compound obtained,        cleavage from the resin, extraction and purification of the        peptide obtained, biotinylated at its amino terminal, the steps        of side chain deprotection and peptide cleavage being liable to        be performed simultaneously or separately, and particularly    -   deprotection of the NH₂-terminal group of the intermediary        group, for instance by means of piperidine,    -   cleavage from the resin for instance with an acid such as        trifluoroacetic acid, in the presence of scavengers such as        ethanedithiol, thioanisole, or anisole,    -   extraction of the peptide with a solvent such as diethylether to        remove most the acid and scavengers, purification, such as with        HPLC to obtain:

Biotin can be conveniently coupled to the free amino-terminus (of anotherwise fully protected peptide chain using also conventionalactivation procedures. Since biotin possesses one carboxyl group and noamino groups, biotin essentially functions as a chain terminator.Preferred activating agents for in situ activation include but are notlimited to benzotriazol-1-yl-oxy-tripyrrolidinophosphoniumhexafluorophosphate (PyBOP),0-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU), andO-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU). The activation procedures employing these and related compoundsare known to those versed in the art of solid phase peptide synthesisand the coupling of biotin does not entail a significant departure fromstandard coupling protocols.

Biotin in a pre-activated form may also be used. EitherN-hydroxysuccinimidobiotin or biotinamidocaproate N-hydroxysuccinimideester are conveniently employed and both are commercially available.This method of coupling has been described by Lobl, T. J., Deibel, M.R., and Yem, A. W., Anal. Biochem. (1988) 170(2):502-511. Followingaddition of the N-terminal biotin, the peptide is cleaved from the resinin the presence of scavengers, the choice of which will depend on theusual considerations of peptide amino acid composition and the nature ofthe protecting groups used.

(16) The carboxy terminal biotinylated peptides involved in the processof the invention can be prepared according to a method which comprises

-   -   coupling of a carboxy-activated form of the intermediary product        as defined above to a cleavable linker attached to the resin,        for instance to obtain the following compound:    -   deprotection of the α amino group of the intermediary compound,        for instance by means of piperidine to obtain:    -    successive addition of the subsequent amino acids AA₁ . . .        AA_(n) duly protected onto    -    to obtain:    -   deprotection of the NH₂-terminal for instance by means of        piperidine,    -   deprotection of the compound obtained, cleavage from the resin,        extraction and purification of the peptide obtained,        biotinylated at its carboxy terminal end, the steps of side        chain deprotection and peptide cleavage being liable to be        performed simultaneously or separately, and particularly    -   deprotection of the NH₂-terminal, for instance by means of        piperldine,    -   cleavage from the resin for instance with trifluoroacetic acid,        in the presence of scavengers such as ethanedithiol, or        thioanisole, or anisole,    -   extraction of the peptide with a solvent such as diethylether to        remove most of the acid and scavengers,    -   purification, such as with HPLC to obtain:

(17) The internally biotinylated peptides can be prepared according to amethod which comprises the following steps:

-   -   addition of successive amino acids duly protected onto the resin        to give:        Fmoc-AA_(n) . . . AA₁-resin,    -   deprotection of the NH₂-terminal,—addition of the intermediary        product:    -    through its COOH onto the NH₂-terminal to obtain:    -   deprotection of the α amino group of the intermediary compound,        for instance by means of piperidine. to obtain:    -   addition of the subsequent amino acids duly protected onto the        resin to give:    -   deprotection of the NH₂ terminal group of the compound obtained,        cleavage from the resin, extraction and purification of the        peptide obtained, biotinylated at its amino-terminal, the steps        of side chain deprotection and peptide cleavage being liable to        be performed simultaneously or separately, and particularly,    -   deprotection of the NH₂-terminus, for instance by means of        piperidine,    -   cleavage from the resin for instance with trifluoroacetic acid,        in the presence of scavengers such as ethanedithiol, or        thioanisole, or anisole,    -   extraction of the peptide with a solvent such as diethylether to        remove most of the acid and scavengers, purification, such as        with HPLC to obtain:

Under certain circumstances, it may prove particularly advantageous tobe able to biotinylate a peptide internally or at its carboxy-terminus.Such instances arise, for example, when the amino acid sequence of apeptide corresponds to the amino-terminal sequence of a protein.Attachment of a biotin to the amino-terminus of such a peptide resultsin a structure which is significantly different from that found in thenative protein and may, as a consequence, adversely affect the bindingproperties of biochemical properties of the peptide. It is also possiblethat even for peptides corresponding to internal protein sequences,their recognition by binding proteins or immunoglobulins may depend onwhich end of the peptide and then manner in which it is presented forbinding. The importance of peptide orientation has been described byDyrberg, T. and Oldstone, M. B. A., J. Exp. Med. (1986) 164:1344-1349.

In order to be able to incorporate a biotinyl moiety into a peptide in aposition and sequence independent manner, efforts were made tosynthesize a suitable reagent which can be coupled using conventionalprocedures. A convenient reagent for C-terminal or internalbiotinylation is N-ε-biotinyl-lysine. Provided the a-amino group of thiscompound is suitably protected (Fmoc and tBoc), this reagent may be usedto introduce a biotin anywhere in the peptide chain, including at theamino terminus, by the standard procedures used in solid phase peptidesynthesis.

The synthesis of the t-Boc-protected derivative has been described(Bodansky, M., and Fagan, D T., J. Am. Chem. Soc. (1977) 99:235-239) andwas used to synthesize short peptides for use in studying the enzymeactivities of certain transcarboxylases.

Unlike the t-Boc derivative, the synthesis of N-α-Fmoc-Lys(N-ε-biotin)has not been described and given the growing interest inFmoc-based synthesis strategies, this compound is consideredparticularly advantageous.

There are a number of possible routes which can be taken to arrive atthe desired Fmoc-protected compound.

These are shown in FIG. 1. In the first approach, commercially availableN-α-Fmoc-Lys (N-ε-tBoc) can be used as the starting material. TheN-ε-tBoc protection is removed using trifluoroacetic acid and ascavenger such as water. A slight molar excess of the N-α-Fmoc-lysine soobtained is then reacted with carboxy-activated biotin. The resultingproduct can be readily purified by selective extractions and standardchromatographic techniques. In an alternative approach, N-α-Fmoc-Lys(N-ε-biotin) can be produced from commercially available N-ε-biotinyllysine (biocytin) by reaction with fluorenylmethylsuccinimidylcarbonate. Numerous examples of these reactions which can be used asguidelines are given in Atherton and Sheppard, Solid Phase PeptideSynthesis, IRL Press, 1989.

The strategy shown in FIG. 1 (method A) may also be applied tosynthesize N-α-Fmoc-ornithine (N-δ-biotin) from commercially availableN-α-Fmoc-ornithine (N-δ-tBoc). The ornithine derivative differs from thelysine derivative only in the length of the side chain which, for theornithine derivative, is shorter by one carbon atom.

The N-α-Fmoc-Lys can be conveniently incorporated into the peptide chainusing the same reagents for in situ activation described for freebiotin.

Alternatively, N-α-Fmoc-Lys (N-ε-biotin)-O-pentafluorophenyl ester canbe conveniently synthesized from N-α-Fmoc-Lys (N-ε-biotin) andpentafluorophenyl trifluoroacetate using the base-catalyzedtransesterification reaction described by Green, 1K. and Berman, J.,Tetrahedron Lett. (1990) 31:5851-5852, for the preparation of0-pentafluorophenyl esters of amino acids. This active ester can be useddirectly to incorporate N-α-Fmoc-Lys(N-ε-biotin) into the peptide chain.The class of above-defined intermediary products can be preparedaccording to a method which comprises the following steps:

-   -   reaction of a diamino-, monocarboxylic acid previously described        with fluorenylmethysuccinimidylcarbonate or fluorenylmethyl        chloroformate under conditions of carefully controlled pH to        give the singly protected N-α-Fmoc derivative,    -   or alternatively, use of commercially available        N-α-Fmoc-protected diamino-monocarboxylic acids when the side        chain amino group is provided with a protecting group which is        different from the Fmoc group used to protect the α-amino group,        the side chain amino group protection being liable to be        selectively removed under conditions which leave the N-α-Fmoc        group intact,    -   purification of the mono-protected        N-α-Fmoc-diamino-monocarboxylic acid derivative by selective        extractions and chromatography,    -   reaction of the derivative obtained with a carboxy-activated        derivative of biotin, such as N-hydroxysuccinimide biotin, to        obtain the (N-α-Fmoc)-(N-y-biotin) derivative which is the        desired intermediary product,    -   purification of the intermediary product by selective        extractions, precipitations, or chromatography.

When the biotinylated peptides used in the process of the invention areto be provided with linker arms, these chemical entities may beconveniently attached to either the N- or C-terminus of a peptidesequence during solid phase synthesis using standard coupling protocols,as long as the amino groups of these compounds are provided withappropriate temporary amino group protection.

All these specific biotinylated peptides are new.

DESCRIPTION OF THE FIGURES

All the samples and sera mentioned in the figures and tables arerandomly chosen samples and sera, containing antibodies produced as aresult of naturally occurring infection by a viral agent.

FIGS. 1 a-1 c represent the strategies for the synthesis ofN-α-Fmoc-lysine (N-ε-Biotin).

More particularly:

Method A corresponds to the synthesis of (N-α-Fmoc-Lys(N-ε-biotin) fromN-ε-Fmoc-Lys(N-ε-tBoc) and Method B corresponds to the synthesis of(N-α-Fmoc-Lys (N-ε-biotin) from N-ε-biotinyl lysine.

FIGS. 2 a and 2 b represent the diagram obtained in reverse phasechromatography of the precursors involved in the preparation of theintermediary products defined above, and of the intermediary compounds.

The reverse phase chromatography has been carried out in the followingconditions:

-   -   gradient specifications:        -   buffer A: 0.1% TFA in H20,        -   buffer B: 0.1% TFA in acetonitrile,        -   column: C2/C1s reverse phase (Pharmacia, Pep-S),        -   detection wavelength: 255 nanometers;    -   gradient:        -   0% B from 0 to 1 minute,        -   0% B to 100% B from 1 minute to 60 minutes,        -   0% B from 60 minutes to 70 minutes.

The first diagram corresponds to method A (see FIG. 1) and the; seconddiagram corresponds to method B (see FIG. 1).

FIGS. 3 a-1 and 3 a-2 represent the antibody binding to HCV peptide II(in an ELISA).

The upper left curve corresponds to sample 8320.

The upper right curve corresponds to sample 8242.

The lower left curve corresponds to sample 8243.

The lower right curve corresponds to sample 8318.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

The curve with crosses corresponds to non-biotinylated HCV peptide IIand the curve with dots corresponds to biotinylated HCV peptide II.

FIGS. 3 b-1 and 3 b-2 represent the antibody binding to HCV peptide XI(in an ELISA).

The upper left curve corresponds to sample 8320.

The upper right curve corresponds to sample 8326.

The lower left curve corresponds to sample 8242.

The lower right curve corresponds to sample 8243.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

The curve with crosses corresponds to non-biotinylated HCV peptide XIand the curve with dots corresponds to biotinylated HCV peptide XI.

FIGS. 3 c-1 and 3 c-2 represent the antibody binding to HCV peptide XVI(in an ELISA).

The upper left curve corresponds to sample 8326.

The upper right curve corresponds to sample 8242.

The lower left curve corresponds to sample 8243.

The lower right curve corresponds to sample 8318.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

The curve with crosses corresponds to non-biotinylated HCV peptide XVIand the curve with dots corresponds to biotinylated HCV peptide XVI.

FIGS. 4 a and 4 b correspond to the detection of biotinylated peptidescoated directly (in an ELISA).

The first curve corresponds to biotinylated HCV peptide II, the secondcurve to biotinylated HCV peptide XI and the third curve to biotinylatedHCV peptide XVI.

In each of these samples, the optical density (at 450 nm) is plottedagainst the coating concentration expressed in μg/ml.

FIGS. 5 a and b represent the structures of N- and C-terminallybiotinylated HIV-1 peptides (hereabove designated by 1a.1) originatingfrom the transmembrane (TM) protein of HIV-1.

FIGS. 6 a-1 through 6 a-5 represent the detection of core epitopes inthe Core region of HCV using overlapping 9-mers (in an ELISA).

The sera used are indicated above each diagram. The ordinates correspondto the optical density at 450 nm.

The abscissae correspond to the sequence of the protein in which thelocation of the epitope (s) is to be determined. For purposes of graphicillustration, the optical density is assigned to the first amino acid inthe respective nine-mer sequences.

FIGS. 6 b-1 through 6 b-5 represent the detection of core epitopes inthe NS4 region of HCV using overlapping 9-mers (in an ELISA).

The sera used are indicated above each diagram. The ordinates correspondto the optical density at 450 nm.

The abscissae correspond to the sequence of the protein in which thelocation of the epitope (s) is to be determined. For purposes of graphicillustration, the optical density is assigned to the first amino acid inthe respective nine-mer sequences.

FIGS. 6 c-1 through 6 c-10 represent the detection of core epitopes inthe NS5 region of HCV using overlapping 9-mers (in an ELISA).

The sera used are indicated above each diagram. The ordinates correspondto the optical density at 450 nm.

The abscissae correspond to the sequence of the protein in which thelocation of the epitope(s) is to be determined.

For purposes of graphic illustration, the optical density is assigned tothe first amino acid in the respective nine-mer sequences.

FIGS. 7 a-1 through 7 a-3 correspond to the positions of biotinylated20-mers with respect to overlapping 9-mers (in an ELISA).

The abscissae corresponds to the protein sequence in which theepitope(s) is to be determined.

FIGS. 7 b-1 through 7 b-3 correspond to the positions of biotinylated20-mers with respect to overlapping 9-mers (in an ELISA).

The abscissae corresponds to the protein sequence in which theepitope(s) is to be determined.

FIGS. 7 c-1 through 7 c-4 correspond to the positions of biotinylated20-mers with respect to overlapping 9-mers (in an ELISA).

The abscissae corresponds to the protein sequence in which theepitope(s) is to be determined.

FIG. 8 represents a comparison of antibody recognition of biotinylatedand unbiotinylated HCV peptides by line immunoassay (LIA).

FIG. 9 represents a comparison of antibody recognition of biotinylatedcore peptides by line immunoassay (LIA).

The shorter and longer peptides are compared.

FIG. 10 represents an evaluation of type-specific HCV NS4 peptides byLine immunoassay (LIA).

FIG. 11 represents the amino acid sequence of peptides NS4-a to NS4-e.

FIG. 12 represents the composition of hybrid HCV peptides.

FIG. 13 represents the antibody recognition of hybrid HCV peptides.

FIGS. 14 a-14 d represent the construction scheme for mixotope peptidesfrom the N-terminus of E2/NS1 of HCV type 1.

FIG. 15 represents the mixotope synthesis strategy.

FIGS. 16A and 16B represent the synthesis of multiple antigen peptides(MAPs).

FIGS. 17A and 17B represent the recognition of E2/NS1 peptides, by serafrom rabbits immunized with E2/NS1 “b” peptide MAPs.

FIG. 18 represents the recognition of a commercially available serumpanel with a number of biotinylated HTLV-I and HTLV-II peptidesincorporated into LIA strips.

Table 1 represents the antibody recognition of unbiotinylated HIV-1 andHIV-2 peptides (designated by TM-HIV1 and TM-HIV-2) and biotinylatedHIV-1 and HIV-2 peptides (hereabove referred to as 1a.1 and 2a, and alsodesignated by TM-HIV-1 Bio and TM-HIV-2 Bio) in an ELISA.

Table 2 represents the comparison of antibody recognition ofunbiotinylated and biotinylated peptides from the V3 sequence of isolateHIV-1 mn (also referred as 1b.4) in an ELISA.

Table 3 represents the comparison of antibody recognition of thebiotinylated V3″mn peptide (referred to as 1b.4) bound to streptavidinand avidin, in an ELISA.

Table 4 represents the comparison of antibody recognition ofbiotinylated and unbiotinylated HCV peptides, in an ELISA.

More Particularly:

Table 4A corresponds to the antibody binding to HCV peptide XI.

Table 4B corresponds to the antibody binding to HCV peptide XVI.

Table 4C corresponds to the antibody binding to HCV peptide II.

Table 4D corresponds to the antibody binding to HCV peptide III.

Table 4E corresponds to the antibody binding to HCV peptide V.

Table 4F corresponds to the antibody binding to HCV peptide IX.

Table 4G corresponds to the antibody binding to HCV peptide XVIII.

Table 5 represents a comparison of antibody binding to biotinylated andnon-biotinylated peptides, at different peptide coating concentrations,in an ELISA.

Table 6 represents the comparison of N- and C-terminally biotinylatedTM-HIV-1 peptide (referred to as 1a.1), in an ELISA.

Table 7 represents a comparison of antibody recognition ofunbiotinylated and carboxy-biotinylated HCV peptide I.

Table 8 represents the use of mixtures of biotinylated HIV and HCVpeptides for antibody detection, in an ELISA.

Table 9 represents sequences of the core epitopes of the HCV Coreprotein.

Table 10 represents sequences of the core epitopes of the HCV NS4protein.

Table 11 represents sequences of the core epitopes of the HCV NS5protein.

Table 12 represents the antibody binding of various Core, NS4, and NS5biotinylated 20-mers by 10 test sera.

Table 13 represents the antibody recognition of individual E2/NS1peptides (percent of all sera giving a positive reaction).

Table 14 represents the overall recognition of HIV V3-loop peptides.

Table 15 represents the recognition of HIV peptides according to thegeographical region.

Table 16 represents the recognition of European, African and BrazilianHIV-1-positive sera to HIV-I V3-loop peptides V3-con and V3-368.

Table 17 represents the recognition of HIV-2 positive sera to two HIV-2V3 loop peptides.

Table 18 represents the antibody recognition of hybrid peptides. Table19 represents the antibody recognition of mixed HTLVI and II, peptides.

All amino acid sequences are given in the conventional and universallyaccepted three-letter code and where indicated in the one-letter code.The peptide sequences are given left to right which, by convention, isthe direction from the amino terminus to the carboxy-terminus.

A number of unconventional codes are also used to represent chemicalgroups or modifications and are defined as follows: Group Code Ac acetylBio D-biotinyl Fmoc 9-fluorenylmethoxycarbonyl tBoc tertiarybutyloxycarbonyl

EXAMPLE 1 Peptide Synthesis

All of the peptides described were synthesized on TentaGel S-RAM (RappPolymere, Tübingen, Germany), a polystyrene-polyoxyethylene graftcopolymer functionalized with the acid-labile linker4-(α-Fmoc-amino-2′,4′-dimethoxybenzyl) phenoxyacetic acid (Rink,Tetrahedron Lett. (1987) 28:3787) in order to generate peptidecarboxy-terminal amides upon cleavage. t-Butyl-based side chainprotection and Fmoc-α-amino-protection was used. The guanidine-group ofarginine was protected with the 2,2,5,7,8-pentamethylchroman-6-sulfonylmoiety. The imidazole group of histidine was protected with either t-Bocor trityl and the sulfhydryl group of cysteine was protected with atrityl group. Couplings were carried out using preformedO-pentafluorophenyl esters except in the case of arginine where TBTU wasused as the activating agent in the presence of 1.5 equivalents of thebase N-methylmorpholine. Occasionally, glutamine and asparagine werealso coupled using TBTU activation. In these cases, the trityl-protectedderivatives of these amino acids were employed. Biotin was coupled usingeither TBTU or HBTU. All syntheses were carried out on a Milligen 9050PepSynthesizer (Novato, Calif.) using continuous flow procedures.Following cleavage with trifluoroacetic acid in the presence ofscavengers and extraction with diethylether, all peptides were analyzedby C18-reverse phase chromatography.

EXAMPLE 2 Synthesis of N-α-Fmoc-Lys (N-ε-biotin)

A. Method A

Commercially available N-α-Fmoc-L-lysine (N-ε-tBoc) (1.5 grams) wastreated with 20 milliliters of 95% trifluoroacetic acid, 5% H₂O for 2hours at room temperature. Most of the acid was then evaporated under astream of nitrogen. Ten milliliters of water was added and the solutionwas extracted 3 times with diethylether. The aqueous phase was thenevaporated to dryness in vacuo over phosphorus pentoxide. The resultingpowder (N-α-Fmoc-L-lysine) was analyzed by reverse phase chromatographyand revealed a homogeneous product which was, as expected, morehydrophilic than the starting material.

N-α-Fmoc-lysine (190 mg, 0.49 mmol) was dissolved in 8 milliliters of0.1 M borate buffer, pH 8.7. N-hydroxysuccinimidobiotin (162 mg, 0.47mmol) was dissolved in 4 milliliters of dimethylformamide and added tothe solution of N-α-Fmoc-lysine. The pH was monitored and titrated asnecessary, with NaOH. After 2 hours, the solution was acidified with HClto pH 2.0, at which time a white precipitate was obtained.

Following extraction with ethylacetate and centrifugation, the whiteprecipitate was found at the H2O: ethylacetate interface. Both phaseswere removed and the precipitate extracted twice with 10 mM HCl, oncewith ethylacetate, followed by two extractions with diethylether. Theprecipitate was dissolved in DMF and precipitated by addition ofdiethylether. The crystalline powder was then dried in vacuo overphosphorus pentoxide. The resulting product was analyzed by reversephase chromatography and revealed a major peak which, as expected,eluted later than N-α-Fmoc-Lys. A very small peak of N-α-Fmoc-Lys wasalso observed. (FIG. 2 a).

B. Method B

Commercially available N-ε-biotinyl lysine (biocytin, Sigma, 249 mg,0.67 mmol) was dissolved in 8 milliliters of 1 M Na₂CO₃ and cooled onice. Fluorenylmethylsuccinimidyl carbonate (222 mg, 0.66 mnol) wasdissolved in 2 milliliters of acetone and was added to the biotinyllysine solution over a period of 30 minutes with vigorous stirring.Stirring was continued for 5 hours at room temperature. The pH wasmaintained between 8 and 9 by addition of 1 M Na₂CO₃ as necessary. Theacetone was then evaporated off under vacuum, and 1.0 M HCl was addeduntil the pH of the solution was approximately 2. Upon acidification ofthe solution, a white precipitate appeared which was washed twice with10 mM HCl, twice with ethyl acetate, and twice with diethylether. Theprecipitate was dissolved in DMF and precipitated by addition ofdiethylether. The crystalline powder was then thoroughly dried in vacuumover phosphorus pentoxide. The resulting product was analyzed by reversephase chromatography and revealed a major peak which eluted with thesame retention time(30.5 minutes) as the product obtained using method1(FIG. 2 b).

EXAMPLE 3 Methods for the Determination of Peptides Corresponding toImmunologically Important Epitopes in an Enzyme-Linked ImmunosorbentAssay (ELISA) Using Specific Antibodies

Where peptides were to be coated directly, stock solutions of thepeptides were diluted in sodium carbonate buffer, pH 9.6 and used tocoat polystyrene microtiter plates at a peptide concentration of 2 to 5micrograms per milliliter for 1 hour at 37° C.

In cases where biotinylated peptides were to be evaluated, plates werefirst coated with streptavidin in sodium carbonate buffer, pH 9.6 at aconcentration of 3 micrograms per milliliter for 1 hour at 37° C. Theplates were then washed to remove excess, unbound protein. A workingsolution of the biotinylated peptide at 1 microgram per milliliter insodium carbonate buffer was then added to the wells of the microtiterplate and incubated for 1 hours at 37° C.

Once the plates had been coated with antigen, any remaining free bindingsites on the plastic were blocked with casein. After washing, a dilutionof the appropriate antisera, usually 1:100, was added to the wells ofthe plates and incubated for 1 hour at 37° C. After washing to removeunbound material, specific antibody binding was detected by incubatingthe plates with goat anti-human immunoglobulin antibodies conjugated tothe enzyme horseradish peroxidase.

Following removal of unbound conjugate by washing, a solution containingH₂O₂ and 3,3′,5,5′-tetramethylbenzidine was added.

Reactions were stopped after a suitable interval by addition of sulfuricacid. Positive reactions gave rise to a yellow color which wasquantified using a conventional microtiter plate reader. Absorbancemeasurements were made at a wavelength of 450 nanometers and all dataare expressed as an optical density value at this wavelength.

EXAMPLE 4 Use of Biotinylated HIV Peptides for the Detection ofHIV-Specific Antibodies

Experiments were performed to evaluate antibody recognition of short, 10amino acid-long, N-acetylated peptides corresponding to other containedwithin the transmembrane proteins of HIV-1 and HIV-2. Direct coating ofthese peptides in the wells of microtiter plates gave very poor resultswhen antibody binding was evaluated in an ELISA. Since it was suspectedthat the peptides did not bind well to the polystyrene solid phase, thepeptides were resynthesized in the same way except that biotin wasattached to the amino terminus of the peptides, separated from thedecamer peptide sequence by three glycine residues whose function it wasto serve as a linker arm. The peptides used for the comparison were asfollows: TM-HIV-1: Ac-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu- (SEQ ID NO:110)Ile-Cys-NH₂ TM-HIV-1 Bio Bio-Gly-Gly-Gly-Ile-Trp-Gly-Cys-Ser- (SEQ IDNO:111) Gly-Lys-Leu-Ile-Cys-NH₂ TM-HIV-2Ac-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln- (SEQ ID NO:112) Val-Cys-NH₂ TM-HIV-2Bio Bio-Gly-Gly-Gly-Ser-Trp-Gly-Cys-Ala- (SEQ ID NO:113)Phe-Arg-Gln-Val-Cys-NH₂

The biotinylated peptides were loaded onto microtiter plates which hadbeen coated with streptavidin. Antibody binding to these peptides wascompared to antibody binding to the unbiotinylated peptides which werecoated directly onto microtiter plates. The results are shown inTable 1. It is evident that the biotinylated peptides from the HIV-1 orHIV-2 transmembrane proteins bound to streptavidin are recognized verywell by antisera from HIV-1 or HIV-2 infected persons respectively. Thisis in contrast to the unbiotinylated versions of these peptides coateddirectly onto the polystyrene plates. Addition control experimentsshowed that the increase in antibody binding was the result of thespecific interaction between the biotinylated peptide and streptavidin,since there was no difference in antibody recognition of thebiotinylated or unbiotinylated peptides when both were coated directlyonto the microtiter plate. Some peptides, particularly ones which are 15amino acids in length or longer, bind sufficiently to the solid phase toallow the detection of specific antibodies which recognize (an)epitope(s) present in the peptide sequence.

To ascertain whether biotinylation would also improve antibodyrecognition of longer peptides, both the biotinylated and unbiotinylatedversions of the partial V3 loop sequence of isolate HIV-1 mn weresynthesized. The sequence and method of synthesis of both peptides wereidentical except at the amino terminus. The unbiotinylated peptide wassimply acetylated whereas in the biotinylated version, two glycineresidues were added as a linker arm to separate the peptide from thebiotinyl moiety.

The sequences of the two peptides used are as follows: unbiotinylated V3mn peptide Ac-Tyr-Asn-Lys-Arg-Lys-Arg-Ile-His- (SEQ ID NO:114)Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr- Thr-Lys-Asn-Ile-Ile-Gly-NH₂,biotinylated V3 mn peptide (peptide 1b.4)Bio-Gly-Gly-Tyr-Asn-Lys-Arg-Lys-Arg- (SEQ ID NO:115)Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Lys-Asn-Ile-Ile-Gly-NH₂.

The unbiotinylated peptide was coated directly onto the wells of apolystyrene microtiter plate while the biotinylated peptide was bound towells which had previously been coated with streptavidin. The resultsshown in Table 2 demonstrate that antibody binding to the biotinylatedpeptide is superior to antibody binding to peptide coated directly ontothe plastic.

EXAMPLE 5 Use of Biotinylated Peptides—Avidin Complexes for AntibodyDetection

Having demonstrated that antibody recognition of this peptide isimproved when the peptide is biotinylated and bound to streptavidin, anadditional experiment was performed to determine whether streptavidincould be substituted by avidin. The results shown in Table 3 indicatethat this is the case and that biotinylated peptides bound to avidin arerecognized very efficiently by specific antibodies.

EXAMPLE 6 Use of Biotinylated HCV Peptides for Detection of HCV SpecificAntibodies

In order to determine whether the enhanced antibody recognition ofbiotinylated peptides was a general phenomenon, a number of additionaltwenty amino acid-long peptides were synthesized which correspond tosequences derived from the hepatitis C virus (HCV) polyprotein. Theamino acid sequences evaluated were as follows: a. HCV peptide XISer-Gln-His-Leu-Pro-Tyr-Ile-Glu- (SEQ ID NO:116)Gln-Gly-Met-Met-Leu-Ala-Glu-Gln- Phe-Lys-Gln-Lys b. HCV peptide XVILeu-Arg-Lys-Ser-Arg-Arg-Phe-Ala- (SEQ ID NO:117)Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg- Pro-Asp-Tyr-Asn c. HCV peptide IIPro-Gln-Arg-Lys-Thr-Lys-Arg-Asn- (SEQ ID NO:118)Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val- Lys-Phe-Pro-Gly d. HCV peptide IIIArg-Asn-Thr-Asn-Arg-Arg-Pro-Gln- (SEQ ID NO:119)Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly- Gln-Ile-Val-Gly e. HCV peptide VThr-Arg-Lys-Thr-Ser-Glu-Arg-Ser- (SEQ ID NO:120)Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro- Ile-Pro-Lys-Val f. HCV peptide IXIle-Ile-Pro-Asp-Arg-Glu-Val-Leu- (SEQ ID NO:121)Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu- Glu-Cys-Ser-Gln g. HCV peptide XVIIIGlu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr- (SEQ ID NO:122)Glu-Pro-Pro-Val-Val-His-Gly-Cys- Pro-Leu-Pro-Pro

In each case, two versions of the peptide were synthesized. In theunbiotinylated version, the peptide was acetylated at the aminoterminus. The biotinylated versions were all N-terminally biotinylated.A linker arm consisting of two glycine residues separated the biotinylmoiety from the amino acids comprising the HCV sequence.

The unbiotinylated peptides were adsorbed onto the wells of polystyrenemicrotiter plates at a concentration of 3 micrograms per milliliter.

The biotinylated peptides were bound at a concentration of 1 microgramper milliliter to streptavidin-coated microtiter plates. Sera known tocontain antibodies to these peptides were used for the evaluation andwere tested at a 20-fold dilution. The results of these comparisons areshown in Table 4, a to g. These results clearly indicate that antibodyrecognition of biotinylated peptides bound to streptavidin is enhancedrelative to that of peptides coated directly onto the wells of themicrotiter plate.

EXAMPLE 7 Influence of Coating Concentration on Antibody Detection

To investigate further the enhanced antibody recognition of biotinylatedHCV peptides bound to streptavidin or avidin as compared to directadsorption on plastic, the influence of peptide coating concentrationwas investigated. Three peptides (HCV peptides II, XI, and XVI) werecoated in concentrations ranging from 10 nanograms per milliliter to 3micrograms per milliliter in a volume of 200 microliters per microtiterplate well. For direct coating, the unbiotinylated versions of thesepeptides were used. The biotinylated versions of these peptides wereused to coat wells to which streptavidin had previously been adsorbed.Sera known to contain antibodies to these peptides were used at adilution of 1 to 100 to evaluate the magnitude of antibody binding.

The numerical results of this experiment are shown in Table 5 and aredepicted graphically in FIG. 3, a-c.

It is evident that with few exceptions, the biotinylated peptide isrecognized very well even at the lowest concentration tested (10nanograms per milliliter, 2 nanograms per well). In many cases, opticaldensity values close to the maximum attainable are observed at a peptideconcentration of only 30 nanograms per milliliter (6 nanograms perwell). In contrast, however, the unbiotinylated peptides adsorbeddirectly onto the plastic are poorly bound by antibody, if at all.

EXAMPLE 8 Influence of Biotinylation of Peptides on Coating Efficiencyof the Peptides on a Solid Phase

To determine if the absence of a signal was due to lack of peptideadsorption when the peptides were coated directly, an additionalexperiment was performed. In this case, the biotinylated versions of thepeptides were coated directly onto the plastic at the sameconcentrations used in the previous experiment for the unbiotinylatedversions. To ascertain whether biotin-labeled peptide was bound, themicrotiter plates were incubated with a streptavidin: horseradishperoxidase conjugate. Since each peptide contains a single biotinylgroup, the resulting optical densities are a measure of the amount ofpeptide bound, although the absolute amount of bound peptide is notknown. The results presented graphically in FIG. 4 demonstrate thatplastic-bound peptide can be detected. As expected, the curves aredifferent for each peptide which is a reflection of their chemicaluniqueness. Two of the peptides, HCV peptides XI and XVI, appear to bindonly weakly to the wells of the polystyrene microtiter plate and thispoor binding is reflected in the low optical density values obtained inthe ELISA. Since the binding of the biotinylated peptides tostreptavidin-coated wells results in very good antibody recognition, itis obvious that poor binding of the peptide to the solid phase is not alimitation when use is made of interaction between biotin andstreptavidin. On the other hand, one of the peptides, HCV peptide II,shows very significant binding to the solid phase, particularly athigher coating concentrations.

However, at no coating concentration did the signal obtained when thepeptide was coated directly ever equal the signal obtained when thebiotinylated peptide was bound to streptavidin. Since even at the lowestconcentration tested, the streptavidin-bound biotinylated versions ofthis peptide clearly gives a positive signal with the antisera tested,the results would seem to indicate either that the direct coating ofthis peptide is extraordinarily inefficient or that other factors areimportant besides the simple binding of peptide to the solid phase.Although difficult to quantify, one of the factors almost certainlyinvolves the manner in which the peptide is bound and available forantibody binding. In the case of peptides coated directly onto the solidphase, it is virtually inevitable that some proportion of the peptidemolecules will interact with the solid phase through amino acid sidechains which are also essential for antibody recognition. These peptidemolecules will therefore be unable to participate in the bindingreaction with antibodies. This problem is not encountered with thebiotinylated peptides which are all bound to the solid phase through theinteraction between biotin and the solid phase-bound streptavidin.

EXAMPLE 9 Use of C-Terminally Biotinylated HIV Peptides for SpecificAntibody Recognition

In order to determine whether the peptides biotinylated at theircarboxy-terminus also give use to enhanced antibody recognition, acarboxy-biotinylated version of the TM-HIV-1 peptide was synthesized.N-α-Fmoc-Lys (N-ε-biotin) prepared by method A as described was coupleddirectly to resin functionalized with the acid labile linker4-(α-Fmoc-amino-2′,4′-dimethoxybenzyl) phenoxyacetic acid after removalof the linker-bound Fmoc group with 20 percent piperidine. The couplingwas performed using a 3-fold molar excess of N-α-Fmoc-Lys (N-ε-biotin)relative to resin functional groups. Carboxyl group activation wasachieved using one equivalent of HBTU, one equivalent of1-hydroxybenzotriazole and 1.5 equivalents of N-methylmorpholine.N-methyl morpholine was dispensed as a 0.6 M solution indimethylformamide containing 40 percent dimethylsulfoxide which wasnecessary to achieve complete dissolution of the N-α-Fmoc-Lys.(N-ε-biotin). Inspection of the Fmoc deprotection peak followingcoupling of the N-α-Fmoc-Lys (N-ε-biotin) indicated that coupling hadproceeded smoothly and efficiently. Two glycine residues were coupled toseparate the biotinyl lysine from the TM-HIV-1 amino acid sequence.Following synthesis of the peptide, the amino terminus was acetylatedwith acetic anhydride. The resulting structure of thecarboxy-biotinylated peptide differs significantly from the peptidebiotinylated at the amino terminus. A comparison of these structures isshown in FIG. 5.

In order to evaluate antibody recognition of these two peptides, thepeptides were bound individually to streptavidin-coated microtiterplates and tested using a panel of antisera from HIV-1 seropositivedonors. The results of this comparison is shown in Table 6. Clearly,antibody recognition of the C-terminally biotinylated peptide comparesvery favorably with that of the N-terminally biotinylated peptide. Theseresults also confirm the utility of the reagent N-α-Fmoc-Lys(N-ε-biotin)for carboxy-terminal biotinylation.

EXAMPLE 10 Comparison of Antibody Recognition of HCV Peptide I, CoatedDirectly (Unbiotinylated) or Bound to Streptavidin-Coated Plated(Carboxy-Terminal Biotinylation)

A similar experiment was performed using a peptide which bindsrelatively well to polystyrene ELISA plates in order to determinewhether the carboxy-biotinylated form of the peptide would result insuperior antibody recognition relative to the unbiotinylated form of thepeptide. The peptide chosen was HCV peptide I, which was synthesized inthe following versions: a. unbiotinylated version:H₂N-Met-Ser-Thr-Ile-Pro-Lys-Pro- (SEQ ID NO:123)Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr- Asn-Arg-Arg-Pro-Gln-CONH₂ b.carboxy-biotinylated version: H₂N-Met-Ser-Thr-Ile-Pro-Lys-Pro- (SEQ IDNO:124) Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr- Asn-Arg-Arg-Pro-Gln-Gly-Gly-Lys(Bio)-CONH₂.

A spacer consisting of two glycine residues was added at thecarboxy-terminus to physically separate the HCV portion of the peptideproper from the Lys(N-ε-Bio) Synthesis was performed on resinfunctionalized with 4-(α-Fmoc-amino-2′,4′-dimethoxybenzyl) phenoxyaceticacid linker in order to generate carboxy-terminal amides upon cleavage.Coupling of the N-α-Fmoc-Lys(N-ε-biotin) to the linker was performedusing a 3-fold molar excess of the intermediate product relative to thelinker. Activation of the N-α-Fmoc-Lys(N-ε-biotin) was achieved usingone equivalent of TBTU, one equivalent of 1-hydroxybenzotriazole, and1.5 equivalents of N-methylmorpholine. The coupling of all other aminoacids was performed according to conventional protocols. Followingcleavage of the peptides in trifluoroacetic acid in the presence of theappropriate scavengers, the peptides were precipitated and extractedwith diethylether.

Unbiotinylated HCV peptide I was coated directly onto the wells of apolystyrene ELISA plate at a concentration of 3 micrograms permilliliter in sodium carbonate buffer, pH 9.6. Biotinylated HCV peptideI was bound to streptavidin-coated wells using a stock solutioncontaining the peptide at a concentration of 1 microgram per milliliter.The resulting plates were then incubated in parallel with a panel ofsera from HCV-seropositive donors. The results of this comparison areshown in Table 7. The biotinylated peptide clearly gives superiorresults relative to the unbiotinylated version of the same sequence. Twoof the sera (8326 and 6244) recognize the biotinylated version of thispeptide far better than the unbiotinylated version. The specificity ofthe antibody reaction is also reflected by the low optical densityvalues obtained for 5 serum samples from uninfected donors (F88, F89,F76, F136, and F6).

EXAMPLE 11 Use of Mixtures of Biotinylated HIV and HCV Peptides

In many cases, the use of mixtures of peptides is required to give thedesired result. Mixtures of peptides may be used for the detection ofantibodies directed against one or more proteins of a single virus, orfor the detection of antibodies directed against proteins of severalviruses in a single, test. Such tests are considered particularlyadvantageous for the screening of blood donations for their suitabilityfor use in transfusions and as a source of blood products. In suchcases, ELISA plates or other solid supports coated with suitablemixtures of peptides may be used to screen samples for the presence ofantibodies to one or more infectious agents whose presence would renderthe sample unsuitable for use. For the diagnosis of specific infectiousagents, appropriate mixtures of peptides are required in order to obtainaccurate determinations. Antibodies to individual viral antigens derivedfrom one or more infectious agents may be individually detected andidentified simultaneously when use is made of test systems in whichindividual peptides or mixtures of peptides are bound to the solid phasebut are physically separated as they are, for example, in the lineimmunoassay, such that individual reactions can be observed andevaluated. Such tests require the use of an appropriate combination ofpeptide mixtures to achieve the desired result.

It is frequently preferable to use mixtures of peptides rather than asingle peptide for the diagnosis of ongoing or past infections. Sinceindividual responses to single epitopes may be quite variable, morereliable results are often obtained when several immunologicallyimportant epitopes are present in the antibody test. However, since eachpeptide is chemically unique, it is frequently difficult to incorporateall of the desired peptides into one test, particularly when thepeptides are to be coated directly onto the solid phase. Not allpeptides are capable of binding to the solid phase and the peptides inthe mixture may also exhibit very different optimal coating conditionsin terms of pH, ionic strength, and buffer composition.

To determine how well biotinylated peptides would function in a mixturewhen bound to streptavidin- or avidin-coated plates, two mixtures weremade of the N-terminally biotinylated versions of the HIV-1 peptidesTM-HIV-1 (hereabove referred to as 1a.1) and V3-mn (hereabove referredto as 1b.4), the HIV-2 peptide TM-HIV-2 (hereabove referred to as 2a),and the hepatitis C virus peptides II, IX, and XVIII. Mixture Acontained each of the six biotinylated peptides at a concentration of 1microgram per milliliter (6 micrograms per milliliter peptide, total)while in mixture B, each peptide was present at a concentration of 0.1microgram per milliliter (0.6 microgram per milliliter peptide, total).The individual peptides were coated at a concentration of 1 microgramper milliliter. For purposes of comparison, mixtures A and B were alsocoated directly onto the wells of a microtiter plate. Samples fromHIV-1, HIV-2, and HCV-seropositive donors were tested and compared tosera from seronegative blood donors. A cut-off absorbance value of 0.250was used to determine whether a reaction was positive or negative.Absorbance values equal or greater than 0.250 were considered positivewhile absorbance values below this value were considered negative. Theresults of this experiment are shown in Table 8. Based on the reactionsto the individual peptides, all of the HCV serum samples were negativefor antibodies to either HIV-1 or HIV-2. One HIV-2 sample(no. 1400) hadantibodies to HCV peptide XVIII. Of the HIV samples tested, there was noindication of crossreactivity and the ELISA based on individual peptidesis specific. Both mixtures A and B gave good results when bound toavidin-coated microtiter plates. As expected, these mixtures wererecognized by HIV-1, HIV-2, and HCV-positive sera but not by sera fromseronegative blood donors. In contrast, when these mixtures were coateddirectly onto the microtiter plates, the results were considerably lesssatisfactory, with many samples giving a reaction which fell below thecut-off value applied. These results serve to illustrate quiteconvincingly the enhanced immunological recognition of biotinylatedpeptides bound to avidin as compared to peptides coated directly ontothe solid phase as well as the advantages of using mixtures of peptidesfor multiple antibody detection.

EXAMPLE 12 Use of Biotinylated Peptides for Mapping of Epitoped inDiagnostically Useful Regions of HCV

It was demonstrated in Example 6 that several diagnostically importantregions of the HCV polyprotein, such as Core, NS4, and NS5, can beidentified using overlapping 20-mer biotinylated peptides. Extensiveserological testing identified the most useful 20-mer biotinylatedpeptides which permitted to develop a line immunoassay utilizing thesebiotinylated peptides. However, it was desirable to know more exactlywhere in these 20 amino acid-long sequences the epitopes were located.One reason is that, if shorter sequences could be identified, it wouldbe possible to make synthetic peptides containing two or three epitopeswithout the peptide becoming prohibitively long.

Epitopes present in a position of the putative HCV proteins were mappedusing the method originally described by Geysen, H. M., Meloen, R. H.,and Barteling, S. J.; Proc. Natl. Acad. Sci. USA (1984) 81:3998-4002.Consecutive peptides nine amino acids in length with an eight amino acidoverlap were synthesized on polyethylene pins derivatized with anon-cleavable linker. This peptide length was chosen because it islarger than the size of typical linear epitopes which are generallybetween 5 and 7 amino acids in length. By synthesizing 9-mers, theprobability that epitopes would be missed was minimized.

The regions in the HCV polyprotein which were scanned contain Coresequences (aa. 1 to 80), NS4 (aa. 1688 to 1755) and NS5 (aa. 2191 to2330). These regions correspond to the previously determined 20-mers:Peptide I to VII (Core 1 to 13), Peptide VIII to XIV (NS4-1 to 9), andpeptide XV to XIX (NS5-13 to 33).

Following synthesis, all peptides were N-acetylated prior to side chaindeprotection in order to remove the unnatural positive charge at theamino terminus.

The peptides were then assayed for their ability to be recognized byantibodies present in sera from HCV seropositive donors. The results ofthese experiments are shown in FIG. 6 a to 6 c. The optical densityvalues shown are the average of duplicate determinations and have beenassigned to the first amino acid of the 9-mer sequence.

The antibody binding profiles for 10 different HCV sera are shown inFIG. 6 a. It is clear that the Core protein of HCV presents well-definedlinear epitopes which are readily stimulated by synthetic peptides. Atleast superficially, most sera appear to give very similar patterns.Closer inspection, however, reveals that there are individualdifferences. The various regions of the HCV Core protein which arerecognized by antibodies are perhaps more properly termed epitopicclusters rather than epitopes as such, since each region is undoubtedlycomposed of several overlapping epitopes which are difficult, if notimpossible, to distinguish using polyclonal sera. An attempt was made toidentify core epitopes in each of the epitopic clusters. Used in thissense, the word “core” refers to the minimal amino acid sequencerecognizable by antibodies. It should be emphasized, however, that aminoacids in addition to the core sequence may improve reactivityparticularly in the case of polyclonal sera. An analysis of the epitopesis given in Table 9. By comparing the reactions of the various sera,subdomains of epitopic clusters could be identified. Some sera reactpredominately with one subdomain and not with others, while other serarecognize all of the subdomains but still allow the subdomains to bedistinguished because each forms a shoulder in the large peak whichdefines that particular epitopic clusters. Table 9 and FIG. 7 a showsthe locations of the core epitopes with respect to the sequences of the20-mers.

The series of 9-mers corresponding to each of the 20-mer Core peptidesare shown in FIG. 7 a together with the placement of each of thesesequences in relation to an antibody recognition profile for one of theantisera tested.

The antigenic profiles for the NS4 protein obtained with the 10 sera areshown in FIG. 6 b. In general, the reaction of these sera with the9-mers was less pronounced than with the peptides from the Core protein.It was, nevertheless, still possible to identify epitopic regions in theN-terminal sequences of the viral NS4 protein. The core sequences ofthese epitopes are analyzed in Table 10 and show their relation to the20-mer synthetic peptides which are diagnostically important in thisregion. The 9-mers corresponding to the different 20-mers are shown inFIG. 7 b together with their placement in relation to an example of anantigenic profile. It can be seen that the 20-mers correspond quite wellto the epitopes in this region.

The portion of the NS5 protein which was scanned corresponds to theregion covered by the 20-mer peptides 13 to 33. The antigenic profilesobtained in this region are shown in FIG. 6 c. Again, an attempt wasmade to define core epitopes and these are listed in Table 11. Littleantibody binding was observed in the amino terminal portion of thissequence. In FIG. 7 c, the 9-mers corresponding to the 20-mer peptidesNS5-21 to NS5-31 are listed and their positions are shown relative toone of the antigenic profiles.

In particular, it is apparent that, the importance of the sequencerepresented by HCV peptide XVI (NS5-27) would be severely underestimatedbased on the results obtained with the overlapping 9-mers. Theimportance of this sequence would also be underestimated ifunbiotinylated HCV peptide XVI (NS5-27) were evaluated in an ELISAfollowing direct coating onto the microtiter plate (see Table 4B).However, the biotinylated version of this peptide when bound tostreptavidin- or avidin-coated plates reveals the presence of a veryimportant epitope which is of diagnostic value.

In contrast to the often weak binding observed with the 9-mers, thebinding with the 20-mers was frequently quite strong (see table 12). Inseveral cases the differences are dramatic. For example, serum 8241 doesnot recognize any of the 9-mers, whereas the binding to the peptidesHCV2 (peptide IX) and HCV5 (peptide XI) is very strong. Moderate bindingwas also observed to the peptide HCV7 (peptide XIII). This would seem toindicate that there is an important structural component to theseepitopes which is present in the 20-mers but which is absent in the9-mers.

EXAMPLE 13 Use of Biotinylated Peptides for Identification of Epitopesin the N-Termius of NS1 Region of HCV Line Immunoassay

Epitopes can also be identified using the line immunoassay (LIA). Ingeneral, unbiotinylated peptides bind better to nylon membranes than topolystyrene ELISA plates. Nevertheless, biotinylated peptides complexedwith streptavidin or avidin give superior results in the lineimmunoassay than do their unbiotinylated counterparts bound directly tothe membrane. In order to illustrate this, unbiotinylated andN-terminally biotinylated versions of HCV peptides XXg-1 and XXg-2 weresynthesized. The unbiotinylated peptides were applied to the membrane asa stock solution containing 100 micrograms per milliliter peptide,whereas the biotinylated peptides were bound to streptavidin and appliedas a stock solution of 100 micrograms per milliliter complex. The amountof biotinylated peptide in the stock solution was thereforeapproximately 10 micrograms per milliliter. Three human IgG controllines were also applied to the strips in order to assist in evaluatingthe intensity of the reactions. Following application of the antigenlines, excess binding sites on the membrane were blocked with casein inphosphate-buffered saline. The membrane was subsequently cut into stripsperpendicular to the direction in which the antigen lines were appliedand the resulting strips were incubated with a panel of sera fromHCV-seropositive donors. Bound antibody was detected visually using goatanti-human IgG antibodies conjugated to the enzyme alkaline phosphataseafter addition of 5-bromo-4-chloro-3-indolylphosphate and Nitro Bluetetrazolium. The results are shown in FIG. 8.

The specificity of the reactions is demonstrated by the absence ofdetectable antibody binding to any of the HCV peptides -by three sera(33, 34, and 35) obtained from HCV-seronegative donors. The reactions ofsera 1 to 32 to the unbiotinylated HCV peptides XX-1 and XX-2 aregenerally absent or exceedingly weak. In contrast, many of the seratested recognized the biotinylated versions of these peptides whencomplexed to streptavidin. The antibody reactions to the biotinylatedpeptides are significantly stronger in spite of the fact that onlyapproximately one-tenth the amount of peptide was present in these stocksolutions compared to the amount present in the stock solutions of theunbiotinylated peptides. The results obtained using the biotinylatedpeptides demonstrate the presence of a diagnostically useful epitope inthese peptide sequences which is not evident when the unbiotinylatedversions of the peptides are used.

A total of 8 sequences spanning the hypervariable N-terminus of the HCVE2-NS1 region (aa 383 to 416 of the HCV polyprotein) of different HCVisolates were chosen for further evaluation. These aligned sequences(one-letter code) are as following: XXa GETYTSGGAASHTTSTLASLFSPGASQ (1)(SEQ ID NO:125) RIQLVNT XXb GHTRVSGGAAASDTRGLVSLFSPGSAQ (2) (SEQ IDNO:126) KIQLVNT XXc GHTRVTGGVQGHVTCTLTSLFRPGASQ (3) (SEQ ID NO:127)KIQLVNT XXd GHTHVTGGRVASSTQSLVSWLSQGPSQ (4) (SEQ ID NO:128) KIQLVNT XXeGDTHVTGGAQAKTTNRLVSMFASGPSQ (5) (SEQ ID NO:129) KIQLINT XXfAETYTSGGNAGHTMTGIVRFFAPGPKQ (6) (SEQ ID NO:130) NVHLINT XXgAETIVSGGQAARAMSGLVSLFTPGAKQ (7) (SEQ ID NO:131) NIQLINT XXhAETYTTGGSTARTTQGLVSLFSRGAKQ (8) (SEQ ID NO:132) DIQLINT

These sequences are derived from isolates described by the followinggroups:

-   (1) Hijikata et al., Biochem. Biophys. Res. Comm. 175:220-228, 1991.-   (2) unpublished results-   (3) Hijikata et al., Biochem. Biophys. Res. Comm. 175:220-228, 1991.-   (4) Kato et al., Proc. Natl. Acad. sci. USA 87:9524-9528, 1990.-   (5) Takamizawa et al., J. Virology 65:1105-1113, 1991.-   (6) Weiner et al , Virology 180:842-848, 1991.-   (7) Okamoto et al., Japan. J. Exptl. Med. 60:167-177, 1990.-   (8) Kremsdorfl et al., Abstract V64, Third International Symposium    on HCV, Strasbourg, France, September, 1991.

Since the sequences are rather long and because secondarystructure—related difficulties were predicted to occur during synthesis,it was decided to split the sequences into two overlapping parts(a=amino acid 383 to 404 and b=amino acid 393 to 416 of the HCVpolyprotein). Subdividing the sequence also allows the position of theepitope to be move accurately defined.

All of the peptides were N-terminally biotinylated, complexed withstreptavidin and used to prepare LIA-strips (data not shown).

When only the LIA-positive samples are considered, the detection rate onthe E2/NS1 peptides was found to be on the order of 90 percent. Thecorrelations between recognition of the E2/NS1 peptides and LIAreactivity as well as the scores for the individual peptides are shownin Table 13. It was also clear from the observed reactions that theprimary epitope in this sequence is located towards the carboxy-terminusof the hypervariable region. There were exceptions to this. Each serumappeared to have its own recognition pattern which underscores theimportance of using a mixture of different sequences if this epitope isto be included as a line in the LIA. It would also appear that eitherthere is a considerable degree of crossreactivity between the type 1aand type 1b sequences, or that most people are doubly infected. It is asimple matter to distinguish between these two possibilities byselectively removing the antibodies, which bind to one sequence, andlooking to see what the effect is on antibody recognition of the othersequences. A number of samples gave a rather weak reaction to one ormore E2/NS1 peptides but were LIA negative. While most probably falsepositive reactions, these sera may also be from people who werepreviously infected but who have resolved the infection.

EXAMPLE 14 Use of Combined HCV Peptides from the Core Region of HCV forthe Detection of Antibodies by LIA

In order to reduce the overall number of peptides in a HCV ELISA or LIA,biotinylated peptides can be synthesize which span other immunologicallyimportant peptides. Examples of such “combined” HCV peptides from thecore protein NS3 region of HCV are given below: Peptide Sequence core 1M S T I P K P Q R K T K R N T N R R P Q (I) (SEQ ID NO:133) core 2P K P Q R K T K R N T N R R P (II) (SEQ ID NO:134) core 3R N T N R R P Q D V K F P G G G Q I V G (III) (SEQ ID NO:135) coreM S T I P K P Q R K T K R N T N R R P Q 123 D V K F P G G G Q I V G (SEQID NO:136) core 6 V G G V Y L L P R R G P R L G V R A T R (IVa) (SEQ IDNO:137) core 7 L P R R G P R L G V R A T R K T S E R S (IV) (SEQ IDNO:138) core 9 T R K T S E R S Q P R G R R Q P I P K V (V) (SEQ IDNO:139) core 10 R S Q P R G R R Q P I P K V R R P E G R (VI) (SEQ IDNO:140) core G G V Y L L P R R G P R L G V R A T R K 7910T S E R S Q P R G R R Q P I P K V R R (SEQ ID NO:141)

All of these peptides have been provided with a Gly-Gly spacers and abiotin at the amino terminus. The peptides were evaluated in a lineimmunoassay experiment (LIA) and compared to the shorter core peptides.The results are shown in FIG. 9. The longer core peptides compare veryfavorably to the shorter peptides and consistently give a more intensereaction. This is could be explained if (i) the longer peptidesincorporate two or more epitopes which were previously spread over twoseparate peptides and/or (2) there is any conformational contributionwhich may be more prominent in the longer peptides.

EXAMPLE 15 Use of Combined HCV Peptides from the NS4 and NS5 Regions ofHCV for the Detection of Antibodies by LIA

Other peptides combine sequences in NS4 and NS5 which are as following:Peptide Sequence NS4-5 S Q H L P Y I E Q G M M L A E Q F K Q K (XI) (SEQID NO:142) NS4-7 L A E Q F K Q K A L S L L Q T A S R Q A (XIII) (SEQ IDNO:143) NS4-57 S Q H L P Y I E Q G M M L A E Q F K Q KA L G L L Q T A S R Q A (SEQ ID NO:144) NS5-25E D E R E I S V P A E I L R K S R R F A (XV) (SEQ ID NO:145) NS5-27L R K S R R F A Q A L P V W A R P D Y N (XVI) (SEQ ID NO:146) NS5-E D E R E I S V P A E I L R K S R R F A 2527 Q A L P V W A R P D Y N(SEQ ID NO:147)

The general advantage in using the longer peptides lies in the fact thattheir use in an ELISA or LIA leaves more space for the incorporation ofother peptides carrying immunologically important epitopes.

EXAMPLE 16 Use of Type-Specific HCV NS4 Peptides for the Detection ofAntibodies by LIA

Equivalent peptides containing HCV type 2 and type 3 NS4 sequences whichcorrespond to the type 1 peptides found to contain epitopes in NS4 weresynthesized. The sequences of these peptides are shown below forcomparison: Peptide Sequence NS4-1L S G K P A I I P D R E V L Y R E F D E (1) (SEQ ID NO:148) NS4-1V N Q R A V V A P D K E V L Y E A F D E (2) (SEQ ID NO:149) NS4-5S Q H L P Y I E Q G M M L A E Q F K Q K (1) (SEQ ID NO:150) NS4-5A S R A A L I E E G Q R I A E M L K S K (2) (SEQ ID NO:151) NS4-7L A E Q F K Q K A L G L L Q T A S R Q A (1) (SEQ ID NO:152) NS4-7I A E M L K S K I Q G L L Q Q A S K Q A (2) (SEQ ID NO:153)

LIA strips were prepared using these nine peptides which weresubsequently incubated with different sera. The results are shown inFIG. 10. Two of the sera which were previously negative on type 1 NS4peptides gave a positive reaction to the type 3 and type 2 peptides.This indicates that it is possible to increase the NS4 detection rateusing these peptides.

EXAMPLE 17 Use of Biotinylated Peptides from the V3 Loop Region of GP120of Different HIV-1 Isolates in a Line Immunoassay for the Detection ofHIV Antibodies

In order to determine the general diagnostic value of the V3 loop regionof gp120, nine peptides derived from this region of nine different HIV-1isolates were synthesized and included in a-LIA. All nine peptides wereprovided with a Gly-Gly spacer and an N-terminal biotin. The alignedpeptides (one-letter amino acid code) sequences are as following: CONNNTRKSIHI--GPGRAFYTTGEII 23 (SEQ ID NO:154) G SF2NNTRKSIYI--GPGRAFHTTGRII 23 (SEQ ID NO:155) G SCNNTTRSIHI--GPGRAFYATGDII 23 (SEQ ID NO:156) G MNYNKRKRIHI--GPGRAFYTTKNII 23 (SEQ ID NO:157) G RFNNTRKSITK--GPGRVIYATGQII 23 (SEQ ID NO:158) G MALNNTRRGIHF--GPGQALYTTG-IV 22 (SEQ ID NO:159) G BHNNTRKSIRIQRGPGRAFVTIGKI- 24 (SEQ ID NO:160) G ELIQNTRQRTPI--GLGQSLYTT-RSR 22 (SEQ ID NO:161) S ANT70QIDIQEMRI--GP-MAWYSMG-IG 21 (SEQ ID NO:162) G

The peptides were mixed with streptavidin in a slight molar excess overbiotin binding sites and the peptide: streptavidin complexes wereseparated from unbound material over Sephadex G-25. Material eluting inthe void volume was used in the preparation of the LIA.

A total of 332 sera were tested which had been obtained from variousgeographical regions. Since it is known that virus strains isolated inEurope or North America exhibit less strain-to-strain variability thanAfrican isolates, geographical differences in the V3-loop sequencerecognition were to be expected. The reactions of the various lines wereevaluated as positive (i) or negative (o), data not shown.

A complete evaluation of the sera is given in Table 14. In total, 307 ofthe 332 sera gave a reaction to at least one peptide on the V3-loop LIA.Those sera which failed to give a reaction to any peptide on the V3-loopLIA were tested by Western Blot to determine whether the sera wereindeed positive for anti-HIV-I antibodies. It was found that 6 sera werein fact negative. The total number of positive sera tested was therefore326. There were, however, 19 sera which contained antibodies to gp120which failed to react with any of the V3-loop LIA, the percentage ofsera giving a positive reaction was, in global terms, 94%. There were,however, significant geographical differences. These differences areshown in Table 15.

The total percentage of sera from the different geographical regionsgiving at least one positive reaction can be summarized as follows:European 100%  African 94% Brazilian 92%Additional evaluations with European samples indicate that thispercentage is, in fact, some what less than 100% (data not shown).African samples which failed to give a reaction in the LIA have not beentested by Western Blot to confirm the presence of other HIV antibodies.

That the European sera would score well was expected. The lower scoreobtained for the African sera was also not totally unexpected, since itis known that there is more viral heterogeneity in Africa. Since V3-loopsequences of African strains of HIV have not been as extensivelycharacterized as the European or North American strains, it is clearthat we either do not have a representative sequence, or that attemptingto characterize African strains in terms of a consensus sequence is notpossible exercise since there is too much sequence divergence. Theresults obtained with the Brazilian sera were unexpected since nothinghas ever been reported concerning HIV variability in Brazil. From theseresults, it appears that the situation in Brazil more closely resemblesthe situation in Africa and not the situation in North America orEurope.

Example 18 Improved Detection of HIV-1 Anti-V3 Domain Antibodies inBrazilian Sera Using a V3 Sequence Derived from a Brazilian Isolate

Brazilian serum samples which failed to recognize any HIV-1 V3 loopsequences present on the previously described LIA strips but which werepositive for antibodies which recognized the HIV-1 gp120 protein onWestern blots were selected for further study. In one of these samples,V3 loop sequences of virus present in the serum sample could beamplified using the polymerase chain reaction using primers derived fromthe more constant regions flanking the hypervariable domain. Theresulting DNA fragment was subsequently cloned and the nucleotidesequence was determined. A peptide corresponding to the deduced aminoacid sequence encoded by this fragment was synthesized and tested forits ability to be recognized by various HIV-1 antibody-positive sera.The sequence of this peptide was as follows: Peptide V3-368: Asn Asn ThrArg Arg Gly Ile His (SEQ ID NO:163) Met Gly Trp Gly Arg Thr Phe Tyr AlaThr Gly Glu Ile Ile Gly

A spacer consisting of two glycine residues was added to the aminoterminus. Thereafter, the resulting N-terminal glycine residue wasbiotinylated. The ability of European, African, and Brazilian HIV-1antibody-positive sera to recognize this peptide was investigated andcompared to the ability of these same sera to recognize the consensussequence peptide in an ELISA. The two peptides were also evaluatedtogether as a mixture. These results are summarized in table 16. Theseresults demonstrate that with sera of European or African origin, theV3-368 peptide does hot result in an increased anti-V3 loop antibodydetection over that which is observed with the V3con peptide. Incontrast, the use of the V3-368 peptide results in a marked improvementin V3 antibody detection with Brazilian sera. Although this peptide isrecognized less frequently than the V3con peptide, the two peptidescomplement each other to raise the detection rate from 83.3 percentusing the V3con peptide alone to 97.2 percent when the two peptides areused together.

EXAMPLE 19 Antibody Recognition of HIV-2 V3 Loop Sequences

The outer membrane glycoprotein of HIV-2 (gp105) is similar to that ofHIV-1 with respect to its organization. Like the gp120 protein of HIV-1,the gp105 protein of HIV-2 consists of domains of variable sequenceflanked by domains of relatively conserved amino acid sequence. In orderto detect antibodies specific for the V3 domain of HIV-2 produced inresponse to infection by this virus, biotinylated peptides weresynthesized corresponding to the V3 sequences of the HIV-2/SIV isolatesGB12 and isolate SIV mm 239 (Boeri, E., Giri, A., Lillo, F. et al.; J.Virol. (1992) 66(7):4546-4550). The sequences of the peptidessynthesized are as follows: V3-GB12: Asn Lys Thr Val Val Pro Ile Thr(SEQ ID NO:164) Leu Met Ser Gly Leu Val Phe His Ser Gln Pro Ile Asn LysV3-239: Asn Lys Thr Val Leu Pro Val Thr (SEQ ID NO:165) Ile Met Ser GlyLeu Val Phe His Ser Gln Pro Ile Asn Asp

Two glycine residues were added at the N-terminus of each peptide toserve as a spacer and a biotin was coupled to the a-amino group of theresulting N-terminal glycine. The peptides were bound to streptavidinand coated in the wells of microwell plates. HIV-2 antibody-positivesera were used to evaluate these two peptides in an ELISA. These resultsare summarized in Table 17. The results clearly demonstrate theusefulness of these two peptide sequences for the diagnosis of HIV-2infection.

EXAMPLE 20 Localization of the Epitope at the Carboxy Terminus of C-100with Biotinylated Peptides

There have been various reports of an epitope located towards thecarboxy-terminal portion of the C-100 protein (EP-A-0 468 527, EP-A-0484 787). Reactivity of certain sera toward this epitope and not toepitopes located within the 5-1-1 fragment could explain why these seragive a positive reaction on C-100 but not to the above-describedpeptides (described in the above-mentioned examples. The fiveoverlapping biotinylated peptides synthesized NS4-a, b, c, d and e areshown in FIG. 11 and cover the carboxy-terminus of C-100 except for thelast three amino acids. LIA strips prepared with these peptides weretested using a series of HCV Ab-positive and negative sera. The resultsof this experiment (data not shown) are summarized below: peptide Nr. ofreactive sera Percentage NS4-a 0 0% NS4-b 2 3% NS4-c 0 0% NS4-d 0 0%NS4-e 16 27%

EXAMPLE 21 Use of Biotinylated Hybrid Peptides Containing Epitopes fromDifferent HCV Proteins

A fine mapping of the epitopes in the immunologically most importantregions of the HCV polyprotein using 9-mers was performed as illustratedin Example 12. Using this information, 3 peptide sequences were devisedwhich consisted of three 9-mer stretches of HCV sequence separated by 2amino acid-long spacers. In general, Gly-Gly, Gly-Ser or Ser-Gly spacerswere used to provide chain flexibility. The arrangement of the epitopesin the three hybrid peptides synthesized and their sequences are shownin FIG. 12. The three peptides were evaluated on a LIA strip. In thefirst evaluation, the sera originally used for the epitope fine mappingexperiments were used since the precise interactions of these sera withthe epitopes is known. These results are shown in FIG. 13 and aresummarized in Table 16. The order in which the epitopes wereincorporated into these three hybrid peptides was arbitrary. It isadvantageous, however, to link the epitopes together in a limited numberof peptide chains rather than attempting to develop a test based onindividual 9-mers. The use of separate 9-mers would rapidly saturate thestreptavidin binding sites on the plate (one biotin binding site/9-mer)whereas incorporating the 9-mers into a limited number of peptides aswas done in these experiments would enable one to bind 3 times as much(one biotin binding site/three 9-mers).

EXAMPLE 22 E2/NS1 “b” Sequence Mixotope Peptides

The results using synthetic peptides (see Examples above) have indicatedthat most HCV seropositive sera contain antibodies directed towards thehypervariable N-terminus of E2/NS1. However, because of thehypervariable nature of this region of the protein, it is necessary touse a rather wide spectrum of sequences in order to detect theseantibodies in an acceptably high percentage of sera. Analysis ofavailable sequences revealed that the observed amino acid substitutionswere not entirely random and that certain amino acids were preferred incertain positions within the sequence. Since the hypervariable sequenceis rather long, this sequence who divided into two overlapping portions(“a” and “b”) to improve the quality of the product and simplify thesynthesis. Subdividing this region also permitted the determination ofthat the portion of this N-terminal segment of the E2/NS1 protein whichwas most frequently recognized by antibodies was located in the regionencompassed by the “b” versions of these sequences. Given the sequenceinformation shown in FIG. 14 a “mixotope” was synthesized which containsat each position all the amino acids found in the naturally occurringisolates examined. The strategy followed in the synthesis of themixotope is depicted in FIG. 15. The strategy for designing mixotopes isreviewed in Gras-massé et al., Peptide Res. (1992) 5:211-216. The resinwas divided into a number of portions equal to the number of amino acidsto be coupled. The coupling reactions were carried out individually soas to avoid problems arising due to differences in coupling kineticsbetween the various amino acids. Following the coupling reactions, theresin portions were pooled and mixed thoroughly. The total number ofvariants obtained for this 23 amino acid-long sequence was +1.147×1010.The increasing number of variants as a function of chain length asmeasured from the carboxy-terminus or amino-terminus is shown in FIG.14. The rationale behind the mixotope approach is that epitopes arecomposed of amino acids whose contribution to antibody binding is notequal. Antibodies may recognize an epitope even though there may be arelatively large number of (generally not random) substitutions incertain positions. In this respect, the antigenic complexity of themixotope should be substantially less than the number of variantscomprising the mixture. For the sake of illustration, if it is assumedthat an average epitope is 6 amino acids in length, it is possible tocalculate the number variants for each successive 6 amino acid longsegment in the sequence. The number of variants as a function ofposition in the sequence is shown in FIG. 14. The actual number offunctional variant sequences will be equal to the number shown for any 6amino acid-long sequence which happens to correspond to an epitope,divided by a degeneracy factor equal to the number if toleratedsubstitutions in each position of the epitope but modified to reflectthe degree to which the particular substitutions are tolerated.Unfortunately, the exact position(s) of the epitope(s) are not known. Itshould be stated explicitly that this is not a random peptide library.Key positions in the total sequence which do not tolerate substitutions,as evidenced by the absence of amino acid variations in naturallyoccurring isolates, are preserved. One disadvantage to this syntheticapproach is that rare amino acid substitutions are overrepresented andwill tend to dilute out the more commonly encountered amino acids. Onthe other hand, the possibility existed that overrepresentation of raresubstitutions might allow the detection of antibodies not detectablewith epitope sequences comprised of more frequently encountered aminoacids. Following completion of the synthesis of the mixotope, allpeptide chains were provided with a (Gly)2 spacer and a biotin tofacilitate immunological evaluation. A multiple antigen peptide (MAP)version of the mixotope may also be synthesized in parallel. One resultof previous studies was that while approximately 90 percent ofHCV-positive sera could be shown to contain anti-E2/NS1 antibodiesdirected against the N-terminal hypervariable region with the 16 “a” and“b” sequences investigated. The apparent lack of these antibodies in theremaining 10 percent of HCV antibody-positive sera could be due to twofactors: 1) these patients fail to produce antibodies against thisportion of E2/NS1, or 2) has not yet been identified the correctsequence with which to detect these antibodies. Based on experimentswith the HIV-1 V3 loop, this latter possibility did not seem at allunrealistic. LIA strips were prepared which contained the 8 “b”sequences previously used in addition to the mixotope. Sera wereselected which previously scored positive on at least one of the eightdefined sequences as well as sera, which scored negative. In total, 60sera were tested, of which 56 previously gave a positive reaction and 4were previously found to be negative. Of the 56 sera, which hadpreviously scored positive, 21 reacted with only one or two of thepeptides on the strip or only gave a very weak reaction. (data notshown) The mixotope was; recognized by approximately one-third of allthe sera tested. The reaction of some sera to the mixotope wassurprisingly strong, however, it may be possible that the collection ofE2/NS1 sequences on which the mixotope was based is not trulyrepresentative. It is expected that the mixotope MAP will elicit theproduction of broad specificity antisera directed against theamino-terminus of E2/NSI.

EXAMPLE 23 Use of Branched HCV N-Terminal E2/NS1 Region PeptidesForraising Antibodies

Several sequences from the N-terminus of E2/NS1 were selected forsynthesis as multiple antigen peptides (MAP's) using the techniquedescribed by Tam (Proc. Natl. Acad. Sci. USA 85:5409-5413,1988). Thestrategy used to synthesize the branched peptides is shown schematicallyin FIG. 16. Rabbits (two for each MAP) were given an initial injectionand were boosted once before blood was drawn for a first evaluation ofantibody production. The antisera were tested on LIA strips containing atotal of 16 E2 peptides (sequences derived from 8 type 1 isolates, “a”and “b” versions of each). Examination of the LIA strips reveals thatthere is considerable cross-reaction between the antibodies raised inthe rabbits and the various E2 peptides on the strips (FIG. 17). Thefact that both “a” and “b” versions can be found which are recognized bythe different antisera indicates that there is at least one epitopelocated in the region where these two versions overlap.

EXAMPLE 24 Diagnosis of HTLV Infection Using Biotinylated SyntheticPeptides

HTLV-I and II are antigenically related members of a family of oncogenicretroviruses. HTLV-I infection has been shown to be associated with twodisease syndromes:

HTLV-I-associated myelopathy/tropical spastic paraparesis (neurologicaldisorders) and adult T-cell leukemia (ATL) In contrast, HTLV-II has notbeen conclusively linked to any known disease syndrome. This virus wasoriginally isolated from a patient with hairy cell leukemia, however, nocausal relationship between HTLV-II infection and the disease statecould be established. Since HTLV-I infection has definitely beendemonstrated to have the potential to result in human disease whileHTLV-II infection has not, it is of clinical interest to be able todifferentiate between these two infectious agents. Since these twoviruses are antigenically highly related, it is difficult todiscriminate between HTLV-I and HTLV-II infections when viral orrecombinant antigens are used for antibody detection. A number ofbiotinylated peptides were synthesized and evaluated for their abilityto detect antibodies raised in response to infection by either HTLV-I orHTLV-II. Some of the peptides were chosen because they contain epitopes,which are highly conserved between HTLV-I and HTLV-II and shouldtherefore be useful reagents for detecting HTLV infection without regardto virus type. Still other peptides were chosen because they containepitopes, which should allow HTLV-I and HTLV-II infections to bediscriminated. The peptides synthesized are as follows: I-gp46-3: BioGly Gly Val Leu Tyr Ser Pro (SEQ ID NO:166) Asn Val Ser Val Pro Ser SerSer Ser Thr Leu Leu Tyr Pro Ser Leu Ala I-gp46-5: Bio Gly Gly Tyr ThrCys Ile Val (SEQ ID NO:167) Cys Ile Asp Arg Ala Ser Leu Ser Thr Trp HisVal Leu Tyr Ser Pro I-gp46-4: Bio Gly Gly Asn Ser Leu Ile Leu (SEQ IDNO:168) Pro Pro Phe Ser Leu Ser Pro Val Pro Thr Leu Gly Ser Arg Ser ArgArg I-gp46-6: Bio Gly Gly Asp Ala Pro Gly Tyr (SEQ ID NO:169) Asp ProIle Trp Phe Leu Asn Thr Glu Pro Ser Gln Leu Pro Pro Thr Ala Pro Pro LeuLeu Pro His Ser Asn Leu Asp His Ile Leu Glu I-p21-2: Bio Gly Gly Gln TyrAla Ala Gln (SEQ ID NO:170) Asn Arg Arg Gly Leu Asp Leu Leu Phe Trp GluGln Gly Gly Leu Cys Lys Ala Leu Gln Glu Gln Cys Arg Phe Pro I-p19: BioGly Gly Pro Pro Pro Pro Ser (SEQ ID NO:171) Ser Pro Thr His Asp Pro ProAsp Ser Asp Pro Gln Ile Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro Gln ValLeu II-gp52-1: Bio Gly Gly Lys Lys Pro Asn Arg (SEQ ID NO:172) Gln GlyLeu Gly Tyr Tyr Ser Pro Ser Tyr Asn Asp Pro II-gp52-2: Bio Gly Gly AspAla Pro Gly Tyr (SEQ ID NO:173) Asp Pro Leu Trp Phe Ile Thr Ser Glu ProThr Gln Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu HisVal Leu Thr II-gp52-3 Bio Gly Gly Tyr Ser Cys Met Val (SEQ ID NO:174)Cys Val Asp Arg Ser Ser Leu Ser Ser Trp His Val Leu Tyr Thr Pro Asn IleSer Ile Pro Gln Gln Thr Ser Ser Arg Thr Ile Leu Phe Pro Ser II-p19: BioGly Gly Pro Thr Thr Thr Pro (SEQ ID NO:175) Pro Pro Pro Pro Pro Pro SerPro Glu Ala His Val Pro Pro Pro Tyr Val Glu Pro Thr Thr Thr Gln Cys Phe

A number of these peptides were used to prepare LIA strips for thedetection of antibodies to HTLV. Several of the peptides, such as I-p19and I-gp46-4, which are derived from regions of the HTLV-I p19 gagprotein and envelope glycoprotein, respectively, are expected to berecognized by antibodies produced as a result of both HTLV-I and HTLV-IIinfection since these sequences are highly homologous in the twoviruses. Others, such as I-gp46-3, I-gp46-6 for HTLV″I, and II-gp52-1,II-gp52-2 and II-gp52-3 for HTLV-II may be useful for detection ofantibodies as well as discrimination. Since there is some homologybetween the HTLV-I and HTLV-II sequences, cross-reactions are to beexpected. Nevertheless, the intensities of the reactions to the variouspeptides should reveal the identity of the virus to which the antibodieswere produced.

An example of LIA strips prepared with a number of the biotinylatedHTLV-I and HTLV-II peptides is shown in figure XXX. The LIA strips wereevaluated using a commercially available serum panel (Boston BiomedicaInc., mixed titer panel, PRP203). The test results are in completeagreement with the analysis provided by distributor. Only one sample(nr. 9) is positive for HTLV-I. Sample nr.12 is detected as positivebecause of the positive reaction to the peptide I-p19. This sample couldnot be differentiated using these peptides, nor could this sample bedifferentiated by any other test used by the distributor of the serumpanel. Sample nr. 11 was found to be negative and all other samples werefound to be positive for HTLV-II. In an additional experiment, an ELISAwas performed using all 10 of the biotinylated HTLV-I and HTLV-IIpeptides. The peptides were complexed with streptavidin individually andthen mixed prior to coating. Some of the samples from the panel used toevaluate the LIA strips were used to evaluate the peptides in the ELISA.These results are shown in table. The ELISA in this configuration cannotbe used to differentiate HTLV-I and -II infections but should identifyHTLV-positive samples in general regardless of virus type. The resultsfurther demonstrate the utility of these peptides for the diagnosis ofHTLV-infection. TABLE 1 Antibody recognition of biotinylated andunbiotinylated HIV-1 and HIV-2 peptides TM-HIV-1 TM- TM-HIV-2 SerumTM-HIV-1 Bio HIV-2 Bio HIV-1 0724 0.174 2.570 0.000 0.000 positive mm0.051 2.579 0.000 0.000 YEMO 0.162 2.357 0.000 0.000 PL 0.000 1.5590.000 0.000 VE 0.052 2.551 0.000 0.000 HIV-2 1400 0.000 0.000 0.0001.982 positive AG 0.000 0.000 0.000 2.323 53-3 0.000 0.000 0.000 2.365Sero- 194 0.000 0.000 0.000 0.000 negative 195 0.000 0.000 0.000 0.000donors 180 0.000 0.005 0.000 0.000 204 0.000 0.001 0.000 0.000

TABLE 2 Comparison of antibody recognition of biotinylated andunbiotinylated peptides from the V3 sequence of isolate HIV-1 mn. Sampleidentity V3-mn V3-mn Bio Negative control 0.063 0.069 Blank 0.053 0.051YS 1.442 2.784 DV 1.314 2.881 VE 1.717 overflow* OOST 6 1.025 2.855 OOST8 1.389 overflow* 3990 1.442 overflow* PL 0.531 2.351 MM 0.791 2.5424436 0.388 2.268 4438 0.736 2.554 266 0.951 2.591 OOST 4 1.106 overflow**Absorbance value greater than 3.000

TABLE 3 Comparison of antibody recognition of the biotinylated V3-mnpeptide bound to streptavidin and avidin Serum Streptavidin Avidin YS1.236 1.721 DV 1.041 1.748 PL 0.222 0.983 3990 1.391 1.854 VE 1.5261.908 4436 0.596 1.519 Control 0.050 0.063

TABLE 4A Comparison of antibody recognition of biotinylated undunbiotinylated HCV peptides. Antibody binding to HCV peptide XI SerumUnbiotinylated peptide XI Peptide XI  2 0.090 1.971  3 0.443 2.086  40.473 1.976  6 0.053 0.518  8 1.275 2.624 10 0.764 2.321 11 0.569 2.37823 0.775 2.503 31 0.497 2.104 77 0.093 0.159 33 0.832 1.857 49 0.5152.180 negative serum 0.053 0.095

TABLEB 4 Antibody binding to HCV peptide XVI Serum Unbiotinylatedpeptide XVI Peptide XVI  1 1.038 2.435.  2 0.616 1.239  6 0.100 1.595  80.29 1.599 10 1.033 2.847 26 0.053 1.522 83 0.912 2.221 88 1.187 2.51989 0.495 1.530 91 0.197 2.169 95 0.109 1.484 99 0.814 2.045 100 0.4741.637 104 0.205 0.942 105 0.313 2.186 110 0.762 1.484 111 0.193 1.465112 0.253 1.084 113 0.833 2.535 116 0.058 1.918 120 0.964 2.332 114760.068 2.197 24758 0.071 0.062 266 0.712 2.262 8247 0.059 0.618 negativeserum 0.063 0.067

TABLE 4C Antibody binding to HCV peptide II Serum Unbiotinylated peptideII Peptide II 8241 0.444 0.545 8242 1.682 2.415 8243 2.181 2.306 82471.518 1.975 8250 0.110 0.357 8271 0.912 1.284 8273 2.468 2.769 82742.700 2.943 8275 1.489 2.030 8276 2.133 2.345 8277 1.771 2.572 82781.907 2.022 negative serum 0.047 0.070

TABLE 4D Antibody binding to HCV peptide III Serum Unbiotinylatedpeptide III Peptide III 8241 1.219 2.066 8242 1.976 2.197 8243 1.8592.368 8247 1.072 2.398 8248 2.742 2.918 8250 2.47 1 2.626 8271 1.4712.066 8272 2.471 2.638 8273 1.543 2.697 8274 2.503 2.905 8275 1.5952.640 8276 1.976 2.674 8277 0.735 2.327 negative serum 0.050 0.06

TABLE 4E Antibody binding to HCV peptide V Serum Unbiotinylated peptideV Peptide V 8272 0.589 1.220 8273 0.294 1.026 8274 1.820 2.662 82751.728 1.724 8276 2.194 2.616 8277 0.770 1.796 8278 1.391 1.746 82840.040 0.757 negative serum 0.047 0.070

TABLE 4F Antibody binding to HCV peptide IX Serum Unbiotinylated peptideIX Peptide IX 8315 2.614 2.672 8316 0.133 0.367 8317 0.855 1.634 83181.965 2.431 8320 0.721 0.896 8321 0.283 0.457 8326 2.219 2.540 negativeserum 0.052 0.005

TABLE 4G Antibody binding to HCV peptide XVIII Serum Unbiotinylatedpeptide XVIII Peptide XVIII 79 1.739 2.105 83 1.121 1.232 88 0.972 1.85889 2.079 2.309 91 2.202 2.132 99 1.253 1.526 104  1.864 1.998 105  1.5222.053 110  1.981 2.065 111  1.363 1.542 112  1.172 1.408 116  1.5341.978 120  1.599 2.031  1 2.523 2.691 33 1.463 1.813 39 0.068 0.213 472.117 2.611 negative serum 0.001 0.001

TABLE 5 Peptideconcentration* 3.0 1.0 0.3 0.1 0.03 0.01 coatingmethod**1 2 1 2 1 2 1 2 1 2 1 2 Unbiotinylated HCV peptide II and HCV peptide IIsample positive 8320 2.718 2.278 2.684 2.163 2.684 2.004 2.718 1.8282.757 1.272 2.519 0.479 8242 1.427 0.539 1.368 0.408 1.365 0.234 1.3990.058 1.481 0.048 1.196 0.051 8243 1.668 1.341 1.652 1.221 1.608 0.8311.639 0.181 1.597 0.057 1.088 0.056 8318 2.016 0.791 1.993 0.626 1.9580.347 2.001 0.181 2.181 0.095 2.002 0.048 sample negative 1747 0.0640.049 0.071 0.046 0.046 0.041 0.045 0.044 0.045 0.043 0.045 0.041 17810.057 0.053 0.055 0.053 0.051 0.045 0.047 0.046 0.049 0.053 0.053 0.046Unbiotinylated HCV peptide IX and HCV peptide IX sample positive 83201.779 0.129 0.802 0.093 1.798 0.122 1.244 0.063 1.007 0.057 0.461 0.0598326 2.284 0.084 2.271 0.068 2.271 0.078 2.284 0.068 2.193 0.051 1.8120.049 8242 0.791 0.059 0.777 0.052 0.795 0.048 0.911 0.046 0.496 0.0470.215 0.049 8243 1.959 0.063 1.953 0.053 1.892 0.051 1.834 0.051 1.4210.051 0.639 0.054 sample negative 1747 0.051 0.046 0.049 0.046 0.0460.044 0.042 0.045 0.044 0.045 0.043 0.045 1781 0.053 0.053 0.051 0.0520.051 0.051 0.047 0.052 0.048 0.049 0.049 0.051 Unbiotinylated HCVpeptide XVIII and HCV peptide XVIII sample positive 8326 2.315 0.0522.331 0.053 2.331 0.053 2.331 0.049 2.219 0.051 1.848 0.051 8242 0.7490.053 0.839 0.049 0.873 0.048 0.946 0.047 1.188 0.049 1.185 0.048 82430.671 0.057 0.627 0.053 0.629 0.054 0.661 0.051 0.611 0.053 0.462 0.0538318 2.391 0.051 2.396 0.045 2.392 0.047 2.409 0.047 2.308 0.047 1.7110.048 sample negative 1747 0.047 0.048 0.042 0.045 0.061 0.046 0.0440.045 0.058 0.044 0.042 0.047 1781 0.053 0.055 0.048 0.054 0.048 0.0510.048 0.051 0.051 0.051 0.045 0.053*in microgramsper milliliter**1. biotinylatedpeptide on streplavidincoated plate 2.unbiotinylatedpeptidecoated directly

TABLE 6 Comparison of N- and C-terminally biotinylated TM-HIV-1 peptide.TM-HIV-1 TM-HIV-1 Serum C-terminal biotin N-terminal biotin HIV positiveVE 2.079 2.240 OOST 6 1.992 2.003 MM 2.097 2.308 0724 2.322 2.291 DV0.903 1.579 PL 1.893 1.849 2049 1.780 2.058 3990 1.959 1.870 4438 1.6221.697 4436 2.190 2.110 OOST 7 1.728 2.027 OOST 8 2.117 2.237 OOST 92.119 2.222 VCM 2.131 2.263 1164 1.865 1.919 1252 2.244 2.356 0369/872.059 2.042 Seronegative 1784 0.000 0.000 blood donors 1747 0.000 0.0001733 0.014 0.000

TABLE 7 HCV peptide I carboxy- biotinylated (bound to HCV peptide Istreptavidin- (coated directly) coated wells) HCV antibody- positivesera 8316 2.394 2.541 8318 2.385 2.404 8320 2.760 2.762 8326 0.525 1.7758329 2.633 2.672 8333 2.143 2.545 8334 2.271 2.549 8336 1.558 2.016 83441.878 2.010 8248 2.042 2.493 8244 0.077 1.399 8243 2.211 2.541 82421.367 2.389 8364 2.705 2.705 8374 1.070 2.151 8378 2.161 2.531 83301.985 2.651 8387 1.427 2.628 HCV antibody- negative sera F88 0.000 0.026F89 0.017 0.001 F76 0.000 0.022  F136 0.006 0.002 F8  0.000 0.000

TABLE 8 Use of mixtures of biotinylated peptides for antibody detection.TM- HCV HCV HCV TM-HIV- HIV-2- peptide peptide peptide Mixture A MixtureB 1-BIO BIO V3-mn-Bio II-BIO IX-BIO XVIII-BIO Mixture A Mixture B DirectDirect Serum Avidin Avidin Avidin Avidin Avidin Avidin Avidin Avidincoating coating HCV 8243 0.108 0.109 0.114 1.430 1.213 0.118 1.590 1.6380.542 0.184 8247 0.042 0.048 0.052 1.356 0.756 0.046 0.840 1.149 0.0490.049 8248 0.043 0.046 0.048 2.287 0.047 0.905 1.859 2.154 0.407 0.0648269 0.053 0.049 0.056 1.213 0.051 1.513 0.923 1.268 0.078 0.067 82900.045 0.047 0.050 0.060 0.048 2.323 1.210 1.761 0.559 0.717 8278 0.0460.045 0.053 1.878 0.074 0.052 1.806 1.944 0.540 0.152 8273 0.053 0.0500.056 2.017 0.053 0.052 2.037 2.113 0.773 0.185 8285 0.134 0.163 0.1431.592 0.270 0.146 1.746 1.822 0.908 0.401 8291 0.048 0.050 0.053 1.5390.052 0.049 1.591 1.809 0.335 0.098 HIV-2 AG 0.054 2.065 0.068 0.0810.064 0.058 1.833 1.880 0.054 0.056 1400 0.051 1.781 0.055 0.121 0.0521.362 1.692 2.031 0.214 0.326 HIV-1 YS 0.046 0.046 2.201 0.048 0.0490.049 2.045 1.845 0.200 0.052 PL 1.974 0.051 1.321 0.052 0.056 0.0521.587 1.776 0.052 0.055 DV 1.329 0.048 2.340 0.047 0.049 0.047 1.9691.742 0.100 0.049 3990 1.602 0.054 2.319 0.054 0.066 0.056 2.217 1.9260.390 0.081 Blood 1785 0.046 0.047 0.048 0.045 0.050 0.047 0.047 0.0490.045 0.049 donor 1794 0.124 0.090 0.091 0.153 0.098 0.104 0.152 0.1610.050 0.058 1784 0.044 0.046 0.046 0.045 0.050 0.047 0.047 0.047 0.0450.048 1782 0.052 0.057 0.059 0.057 0.062 0.053 0.057 0.059 0.049 0.056

TABLE 9 SEQUENCES OF THE CORE EPITOPES OF THE HCV CORE PROTEIN HCV COREPROTEIN AMINO ACIDS 1-90 POSITIONS OF CORE EPITOPES Epitope 1A: SEQ IDS(181-184)

Epitope 1B: SEQ IDS (185-188)

Epitope 2: SEQ IDS (194-199)

Epitope 3A: SEQ IDS (209-212)

Epitope 3B: SEQ IDS (213-215)

Epitope 3C: SEQ IDS (218-220)

Epitope 4A: SEQ IDS (231-233)

Epitope 4B: SEQ IDS (236-241)

Epitope 5A: SEQ IDS (248-253)

Epitope 5B: (minor) SEQ IDS (254-255)

SEQUENCES OF THE CORE EPI- TOPES OF THE HCV CORE PROTEIN HCV COREPROTEIN AMINO ACIDS 1-90 POSITIONS OF CORE EPITOPES (SEQ ID NO:) Epitope1A: (453) CORE 1 SEQ IDS (454) CORE 2 (181-184) Epitope 1B: (453) CORE 1SEQ IDS (454) CORE 2 (185-188) Epitope 2: (454) CORE 2 SEQ IDS (455)CORE 3 (194-199) Epitope 3A: (456) CORE 5 SEQ IDS (209-212) Epitope 3B:(456) CORE 5 SEQ IDS (457) CORE 7 (213-215) Epitope 3C: (457) CORE 7 SEQIDS (218-220) Epitope 4A: (458) CORE 9 SEQ IDS (231-233) Epitope 4B:(458) CORE 9 SEQ IDS (459) CORE 11 (236-241) Epitope 5A: (459) CORE 11SEQ IDS (600) CORE 13 (248-253) Epitope 5B: (600) CORE 13 (minor) SEQIDS (254-255)

TABLE 10 SEQUENCES OF THE CORE EPITOPES OF THE HCV NS4 PROTEIN HCV NS4PROTEIN Position of core epitopes Epitope 1: SEQ IDS (258-268)

Epitope 2A: SEQ IDS (281-285)

Epitope 2B: (minor) SEQ IDS (286-292)

Epitope 3A: SEQ IDS (293-295)

Epitope 3B: SEQ IDS (296-300)

Epitope 4: SEQ IDS (301-317)

SEQUENCES OF THE CORE EPITOPES OF THE HCV NS4 PROTEIN HCV NS4 PROTEINPositions of core epitopes (SEQ ID NO:) Epitope 1: (460) HCV1 SEQ IDS(461) HCV2 (258-268) Epitope 2A: (462) HCV3 SEQ IDS (463) HCV4 (281-285)(464) HCV5 Epitope 2B: (463) HCV4 (minor) (464) HCV5 SEQ IDS (286-292)Epitope 3A: (464) HCV5 SEQ IDS (465) HCV6 (293-295) (466) HCV7 Epitope3B: (465) HCV6 SEQ IDS (466) HCV7 (296-300) Epitope 4: (466) HCV7 SEQIDS (467) HCV8 (301-317)

TABLE 11 SEQUENCES OF THE CORE EPITOPES OF THE HCV NS5 PROTEIN HCV NS5PROTEIN Positions of the core epitopes Epi- tope 1A: SEQ IDS (348-349)

Epi- tope 1B: SEQ IDS (350-352)

Epi- tope 2: SEQ IDS (360-363)

Epi- tope 3: SEQ IDS (366-370)

Epi- tope 4: (minor) SEQ IDS (374-377)

Epi- tope 5: SEQ IDS (378-385)

Epi- tope 6: SEQ IDS (387-400)

SEQUENCES OF THE CORE EPITOPES OF THE HCV NS5 PROTEIN HCV NS5 PROTEINPositions of the core epitopes Epi- NS5-25 tope 1A: SEQ IDS (348- 349)Epi- NS5-25 tope 1B: SEQ IDS (350- 352) Epi- NS5-27 ttope 2: SEQ IDS(360- 363) Epi- NS5-29 tope 3: SEQ IDS (366- 370) Epi- NS5-29 tope 4:(minor) SEQ IDS (374- 377) Epi- NS5-31 tope 5: SEQ IDS (378- 385) Epi-NS5-31 tope NS5-33 6: SEQ IDS (387- 400)

TABLE 12 Antibody binding of various Core, NS4, and NS5 biotinylated20-mers by the 10 test sera PEPTIDE ELISA (O.D.) SERUM Core-2 Core-3Core-7 Core-9 HCV-2 HCV-5 HCV-7 NS5-25 NS5-27 NS5-31 8242 2.415 2.1970.632 2.315 2.114 1.625 1.252 0.268 2.318 2.453 8248 2.441 2.918 1.5292.821 0.142 0.182 1.963 0.054 0.388 1.511 8332 1.977 2.054 1.387 1.4550.392 0.575 0.945 0.047 2.130 2.290 8339 2.030 2.765 0.166 2.698 2.4970.043 0.041 1.495 2.359 2.757 8358 1.982 2.135 0.357 0.685 1.779 0.6230.598 0.069 2.249 0.182 8377 2.181 2.368 0.221 0.076 2.368 2.227 1.8291.092 2.336 1.378 8378 2.140 2.369 1.089 1.228 1.859 1.449 2.006 0.2791.602 2.337 8383 2.463 2.463 0.970 2.162 2.300 1.018 2.504 0.055 2.3902.358 8241 0.545 2.066 0.448 0.274 2.421 2.280 0.968 0.050 2.456 0.2738243 2.306 2.368 1.251 1.378 2.203 2.268 2.251 0.062 1.444 0.127

TABLE 13 Antibody recognition of individual E2/NS1 peptides (percent ofall sera giving positive reaction) CL14-A  7(51)  13.7% B 36(51)  70.6%KATO-A  2(51)  3.92% B 26(51) 50.98% HCJ4-A 32(51) 62.74% B 41(51)80.39% FRENCH-A 30(51) 58.8% B 42(51) 82.35% YEK-A  7(51) 13.72% B49(51) 96.07% TAMI-A 12(51) 23.52% B 36(51) 70.58% I8CH1-A  5(51)  9.8%B 30(51) 58.82% CHIR-A 32(51)  62.7% B 40(51) 78.42%

TABLE 14 Overall Recognition of V3-Loop Peptides CON SC MN SF2 BH RF MALELI 70 Total SUM 287 261 275 258 108 146 140 24 6 COUNT 326 326 326 326326 326 326 326 326 Gp120 positive % Reactive 88 80 84 79 33 45 43 7 2Total SUM 287 261 275 258 108 146 140 24 6 COUNT 307 307 307 307 307 307307 307 307 HIV-V3 positive % Reactive 93 85 90 84 35 48 46 8 2

TABLE 15 Recognition of Peptides According to Giographical RegionEUROPEAN % AFRICAN % BRAZILIAN % Consensus 98 Consensus 89 Consensus 82HIV-1 (SC) 98 HIV-1 (MN) 85 HIV-1 (MN) 78 HIV-1 (SF2) 98 HIV-1 (SF2) 79HIV-1 (SC) 75 HIV-1 (MN) 97 HIV-1 (SC) 73 HIV-1 (SF2) 72 HIV-1 (RF) 75HIV-1 (MAL) 60 HIV-1 (RF) 38 HIV-1 68 HIV-1 (RF) 34 HIV-1 (MAL) 30 (MAL)HIV-1 (IIIB) 61 HIV-1 (IIIB) 27 HIV-1 (IIIB) 26 HIV-1 (ELI) 8 HIV-1(ELI) 13 HIV-1 (ELI) 5 ANT 70 2 ANT 70 2 ANT 70 2

TABLE 16 Recognition of European, African and Brazilian HIV-1 antibody-positive sera to HIV-1 V3 loop peptides V3-con and V3-368 V3-con V3-368V3con + V3-368 European sera number tested 36 36 36 number 33 4 33positive number 0 12 0 negative number 3 20 3 borderline percent 92 1192 positive percent 0 33 0 negative percent 8 56 8 borderline Africansera number tested 45 45 45 number 40 5 40 positive number 5 31 2negative number 1 9 3 borderline percent 89 11 89 positive percent 9 694 negative percent 2 20 7 borderline Brazilian sera number tested 36 3636 number 30 16 35 positive number 1 5 1 negative number 5 15 0borderline percent 83.3 44.4 97.2 positive percent 2.8 13.9 2.8 negativepercent 13.9 41.7 0 borderline

TABLE 17 Recognition of HIV-2 positive sera to peptides from the V3 loopregion of HIV-2 V3-GB12 V3-239 number tested 21 21 number positive 21 19number negative 0 0 number borderline 0 2 percent positive 100 90.50percent negative 0 0 percent borderline 0 9.5

TABLE 18 Antibody recognition of hybrid peptides Discrepancies A. NS4NS5 Core LIA Serum Epitope 1 Epitope 5 Epitope 2 Epi-152 8241 + − − +(weak) 8242 + − + + 8243 + − + + 8248 + − + + 8332 + + + + 8339 + + + +8358 + − + + 8377 + − + + 8378 + + + + 8383 + + + + B. NS5 NS4 Core LIASerum Epitope 3 Epitope 3B Epitope 3A Epi-33B3A 8241 − − + − 8242− + + + 8243 − + + + 8248 − − + + 8332 + +/− + + 8339 − − + + 8358 + − +− 8377 − + + − 8378 + + − + 8383 + + + + C. Core NS4 NS5 LIA SerumEpitope 4B Epitope 2A Epitope 6 Epi-4B2A6 8241 − − − − 8242 + − − + 8243− +/− − − 8248 + − − + 8332 + +/− − + (weak) 8339 + − + + 8358 − − − +/−8377 − + − − 8378 + + − + 8383 + + − +

TABLE 19 ANTIBODY RECOGNITION OF HTLV PEPTIDES Serum number Opticaldensity 1 0.303 2 3.001 3 0.644 4 1.262 6 3.001 7 2.623 9 2.607 (HTLV-I)10 3.001 11 0.058 (negative) 13 3.001 14 3.001 15 0.850 16 0.278 191.048 20 3.001 21 0.805 22 0.812 23 3.001 24 0.405 25 1.521

1. A peptide consisting of an amino acid sequence selected from SEQ IDNO:455 and SEQ ID NO:459: (A)-GHTRVTGGVQGHVTCTLTSLFRPGASQKIQ (SEQ ID NO455) LVNT-(Z) (A)-AETIVSGGQAARAMSGLVSLFTPGAKQNIQ (SEQ ID NO 459)LINT-(Z),

and, wherein A, when present, represents an amino acid, amino group, orchemically modified amino terminus of the peptide, and wherein Z, whenpresent, represents an amino acid, OH-group, NH₂-group, or a linkageinvolving an OH-group or an NH₂-group or a peptide fragment consistingof at least 5 amino acids of SEQ ID NO: 455 or SEQ ID NO:459 which isimmunologically reactive with HCV antisera.
 2. A peptide consisting ofan amino acid sequence selected from SEQ ID NO:455 and SEQ ID NO:459:(A)-GHTRVTGGVQGHVTCTLTSLFRPGASQKIQ (SEQ ID NO 455) LVNT-(Z)(A)-AETIVSGGQAARAMSGLVSLFTPGAKQNIQ (SEQ ID NO 459) LINT-(Z),

wherein A, when present, represents an amino acid, amino group, orchemically modified amino terminus of the peptide, and wherein Z, whenpresent, represents an amino acid, OH-group, NH₂-group, or a linkageinvolving an OH-group or an NH₂-group; or a peptide fragment consistingof at least 5 amino acids of SEQ ID NO: 455 or SEQ ID NO:459 which isimmunologically reactive with HCV antisera; and said peptide or peptidefragment containing at least one N-terminal biotin group, C-terminalbiotin group or biotin group attached to an internal amino acid; saidbiotin group being attached directly to the peptide or peptide fragmentor attached to the peptide or peptide fragment through a linker Y; saidlinker Y consisting of 1 to 10 chemical entities selected from the groupconsisting of a glycine residue, beta-alanine, 4-aminobutyric acid,5-aminovaleric acid and 6-aminohexanoic acid.
 3. A peptide or peptidefragment of claim 1 or 2 wherein at least one of A and Z are notpresent.
 4. A solid phase comprising a peptide or peptide fragmentaccording to any one of claims 1 to 3 and a solid support wherein thepeptide is anchored to the solid support via at least one covalent ornon-covalent bond.
 5. The solid phase according to claim 3, wherein saidpeptide or peptide fragment is anchored via a biotin group tostreptavidin present on said solid support.
 6. The solid phase accordingto claim 3 or 4 wherein said solid support is a nylon membrane.
 7. Animmunological assay kit for detecting antibodies to HCV comprising atleast one peptide or peptide fragment according to any one of claims 1to
 3. 8. The immunological assay kit according to claim 7 which is aline immunoassay kit.
 9. A peptide fragment according to claim 2consisting of an amino acid sequence (A)-GHTRVTGGVQGHVTCTLTSLFR-(Z) (SEQID NO 465)

, wherein A, when present, represents an amino acid, amino group, orchemically modified amino terminus of the peptide, and wherein Z, whenpresent, represents an amino acid, OH-group, NH₂-group, or a linkageinvolving an OH-group or an NH₂-group or a peptide fragment consistingof at least 5 amino acids of SEQ ID NO: 465 which is immunologicallyreactive with HCV antisera.
 10. A peptide fragment according to claim 2consisting of an amino acid sequence of (A)-GHVTCTLTSLFRPGASQKIQLVNT-(Z)(SEQ ID NO 466)

, wherein A, when present, represents an amino acid, amino group, orchemically modified amino terminus of the peptide, and wherein Z, whenpresent, represents an amino acid, OH-group, NH₂-group, or a linkageinvolving an OH-group or an NH₂-group; or a peptide fragment consistingof at least 5 amino acids of SEQ ID NO: 466 which is immunologicallyreactive with HCV antisera; and said peptide or peptide fragmentcontaining at least one N-terminal biotin group, C-terminal biotin groupor biotin group attached to an internal amino acid; said biotin groupbeing attached directly to the peptide or peptide fragment or attachedto the peptide or peptide fragment through a linker Y; said linker Yconsisting of 1 to 10 chemical entities selected from the groupconsisting of a glycine residue, beta-alanine, 4-aminobutyric acid,5-aminovaleric acid and 6-aminohexanoic acid.
 11. A peptide fragmentaccording to claim 2 consisting of an amino acid sequence(A)-AETIVSGGQAARAMSGLVSLFT-(Z) (SEQ ID NO 473)

, wherein A, when present, represents an amino acid, amino group, orchemically modified amino terminus of the peptide, and wherein Z, whenpresent, represents an amino acid, OH-group, NH₂-group, or a linkageinvolving an OH-group or an NH₂-group or a peptide fragment consistingof at least 5 amino acids of SEQ ID NO: 473 which is immunologicallyreactive with HCV antisera.
 12. A peptide fragment according to claim 2consisting of an amino acid sequence of (A)-ARAMSGLVSLFTPGAKQNIQLINT-(Z)(SEQ ID NO 474)

, wherein A, when present, represents an amino acid, amino group, orchemically modified amino terminus of the peptide, and wherein Z, whenpresent, represents an amino acid, OH-group, NH₂-group, or a linkageinvolving an OH-group or an NH₂-group; or a peptide fragment consistingof at least 5 amino acids of SEQ ID NO: 474 which is immunologicallyreactive with HCV antisera; and said peptide or peptide fragmentcontaining at least one N-terminal biotin group, C-terminal biotin groupor biotin group attached to an internal amino acid; said biotin groupbeing attached directly to the peptide or peptide fragment or attachedto the peptide or peptide fragment through a linker Y; said linker Yconsisting of 1 to 10 chemical entities selected from the groupconsisting of a glycine residue, beta-alanine, 4-aminobutyric acid,5-aminovaleric acid and 6-aminohexanoic acid.
 13. The peptide fragmentaccording to any one of claims 9 to 12 wherein at least one of A and Zare not present.
 14. A solid phase comprising the peptide fragmentaccording to any one of claims 9 to 13 and a solid support wherein thepeptide is anchored to the solid support via at least one covalent ornon-covalent bond.
 15. The solid phase according to claim 14, whereinsaid peptide or peptide fragment is anchored via a biotin group tostreptavidin present on said solid support.
 16. The solid phaseaccording to claim 14 or 15 wherein said solid support is a nylonmembrane.
 17. An immunological assay kit for detecting antibodies to HCVcomprising at least one peptide or peptide fragment according to any oneof claims 9 to
 13. 18. The immunological assay kit according to claim 17which is a line immunoassay kit.